Processes for preparation of 9,11-epoxy steroids and intermediates useful therein

ABSTRACT

Novel 9α-hydroxy steroid compounds, including compounds of Formula IX                    
     wherein —A—A—, —B—B—, R 1 , R 3 , R 8  and R 9  are as defined in the specification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/583,158 filed May 30, 2000, now U.S. Pat. No. 6,335,441, which isa divisional application of U.S. application Ser. No. 09/246,908 filedFeb. 9, 1999, now U.S. Pat. No. 6,180,780, which is a divisionalapplication of U.S. application Ser. No. 08/763,910 filed Dec. 11, 1996,now U.S. Pat. No. 5,981,744, which claims priority from U.S. ProvisionalApplication Serial No. 60/008,455 filed Dec. 11, 1995.

BACKGROUND OF THE INVENTION

This invention relates to the novel processes for the preparation of9,11-epoxy steroid compounds, especially those of the 20-spiroxaneseries and their analogs, novel intermediates useful in the preparationof steroid compounds, and processes for the preparation of such novelintermediates. Most particularly, the invention is directed to novel andadvantageous methods for the preparation of methyl hydrogen9,11α-epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-lactone(eplerenone; epoxymexrenone).

Methods for the preparation of 20-spiroxane series compounds aredescribed in U.S. Pat. No. 4,559,332. The compounds produced inaccordance with the process of the '332 patent have an open oxygencontaining ring E of the general formula:

in which

—A—A— represents the group —CH₂—CH₂— or —CH═CH—,

R¹ represents an α-oriented lower alkoxycarbonyl or hydroxycarbonylradical.

—B—B— represents the group —CH₂—CH₂— or an α- or β-oriented group

R⁶ and R⁷ being hydrogen

X represents two hydrogen atoms or oxo,

Y¹ and y² together represent the oxygen bridge —O—, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X represents H₂, also loweralkanoyloxy,

and salts of such compounds in which X represents oxo and Y² representshydroxy, that is to say of corresponding 17β-hydroxy-21-carboxylicacids.

U.S. Pat. No. 4,559,332 describes a number of methods for thepreparation of epoxymexrenone and related compounds of Formula IA. Theadvent of new and expanded clinical uses for epoxymexrenone create aneed for improved processes for the manufacture of this and otherrelated steroids.

SUMMARY OF THE INVENTION

The primary object of the present invention is the provision of improvedprocesses for the preparation of epoxymexrenone, other 20-spiroxanes andother steroids having common structural features. Among the particularobjects of the invention are: to provide an improved process thatproduces products of Formula IA and other related compounds in highyield; the provision of such a process which involves a minimum ofisolation steps; and the provision of such a process which may beimplemented with reasonable capital expense and operated at reasonableconversion cost.

Accordingly, the present invention is directed to a series of synthesisschemes for epoxymexrenone; intermediates useful in the manufacture ofeplerenone; and syntheses for such novel intermediates.

The novel synthesis schemes are described in detail in the Descriptionof Preferred Embodiments. Among the novel intermediates of thisinvention are those described immediately below.

A compound of Formula IV corresponds to the structure:

wherein:

—A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxy carbonyl, cyano, aryloxy,

R¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical,

R² is an 11α- leaving group the abstraction of which is effective forgenerating a double bond between the 9- and 11-carbon atoms;

—B—B— represents the group —CHR⁶—CHR⁷— or an alpha- or beta-orientedgroup:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring.

A compound of Formula IVA corresponds to Formula IV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula IVB corresponds to Formula IVA wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae IVC, IVD and IVE, respectively, correspond to anyof Formula IV, IVA, or IVB wherein each of —A—A— and —B—B— is —CH₂—CH₂—,R³ is hydrogen, and R¹ is alkoxycarbonyl, preferably methoxycarbonyl.Compounds within the scope of Formula IV may be prepared by reacting alower alkylsulfonylating or acylating reagent, or a halide generatingagent, with a corresponding compound within the scope of Formula V.

A compound of Formula V corresponds to the structure:

wherein —A—A—, —B—B—, R¹, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VA corresponds to Formula V wherein R⁸ and R⁹ withthe ring carbon to which they are attached together form the structure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VB corresponds to Formula VA wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae VC, VD and VE, respectively, correspond to any ofFormula V, VA, or VB

wherein each of —A—A— and —B—B— is —CH₂—CH₂—, R³ is hydrogen, and R¹ isalkoxycarbonyl, preferably methoxycarbonyl. Compounds within the scopeof Formula V may be prepared by reacting an alkali metal alkoxide with acorresponding compound of Formula VI.

A compound of Formula VI corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIA corresponds to Formula VI wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIB corresponds to Formula VIA wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae VIC, VID and VIE, respectively, correspond to anyof Formula VI, VIA, or VIB wherein each of —A—A— and —B—B— is —CH2—CH₂—,and R³ is hydrogen. Compounds of Formula VI, VIA, VIB and VIC areprepared by hydrolyzing a compound corresponding to Formula VII, VIIA,VIIB or VIIC, respectively.

A compound of Formula VII corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIIA corresponds to Formula VII wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIIB corresponds to Formula VIIA wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae VIIC, VIID and VIIE, respectively, correspond toany of Formula VII, VIIA, or VIIB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. A compound within the scope of FormulaVII may be prepared by cyanidation of a compound within the scope ofFormula VIII.

A compound of Formula VIII corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula VIIIA corresponds to Formula VIII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula VIIIB corresponds to Formula VIIIA wherein R⁸ andR⁹ together form the structure of Formula XXXIII:

Compounds of Formulae VIIIC, VIIID and VIIIE, respectively, correspondto any of Formula VIII, VIIIA, or VIIIB wherein each of —A—A— and —B—B—is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of FormulaVIII are prepared by oxidizing a substrate comprising a compound ofFormula XXX as described hereinbelow by fermentation effective forintroducing an 11-hydroxy group into the substrate in α-orientation.

A compound of Formula XIV corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XIVA corresponds to Formula XIV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XIV corresponds to Formula XIVA wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure of Formula XXXIII:

Compounds of Formulae XIVC, XIVD and XIVE, respectively, correspond toany of Formula XIV, XIVA, or XIVB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of Formula XIVcan be prepared by hydrolysis of a corresponding compound within thescope of Formula XV.

A compound of Formula XV corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XVA corresponds to Formula XV wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XVB corresponds to Formula XVA wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure of Formula XXXIII:

Compounds of Formulae XVC, XVD and XVE, respectively, correspond to anyof Formula XV, XVA, or XVB wherein each of —A—A— and —B—B— is —CH₂—CH₂—,and R³ is hydrogen. Compounds within the scope of Formula XV can beprepared by cyanidation of a corresponding compound within the scope ofFormula XVI.

A compound of Formula XXI corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIA corresponds to Formula XXI wherein R⁸ and R⁹together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIB corresponds to Formula XXIA wherein R⁸ and R⁹together form the structure of Formula XXXIII:

Compounds of Formulae XXIC, XXID and XXIE, respectively, correspond toany of Formula XXI, XXIA, or XXIB wherein each of —A—A— and —B—B— is—CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of Formula XXImay be prepared by hydrolyzing a corresponding compound within the scopeof Formula XXII.

A compound of Formula XXII corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIIA corresponds to Formula XXII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIIB corresponds to Formula XXIIA wherein R⁸ andR⁹ together form the structure of Formula XXXIII:

Compounds of Formulae XXIIC, XXIID and XXIIE, respectively, correspondto any of Formula XXII, XXIIA, or XXIIB wherein each of —A—A— and —B—B—is —CH₂—CH₂—, and R³ is hydrogen. Compounds within the scope of FormulaXXII May be prepared by cyanidation of a compound within the scope ofFormula XXIII.

A compound of Formula XXIII corresponds to the structure:

wherein —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined in Formula IV.

A compound of Formula XXIIIA corresponds to Formula XXIII wherein R⁸ andR⁹ together with the ring carbon to which they are attached form thestructure:

where X, Y¹, Y² and C(17) are as defined above.

A compound of Formula XXIIIB corresponds to Formula XXIIIA wherein R⁸and R⁹ together form the structure of Formula XXXIII:

Compounds of Formulae XXIIIC, XXIIID and XXIIIE, respectively,correspond to any of Formula XXIII, XXIIIA, or XXIIIB wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula XXIII can be prepared by oxidation of a compound ofFormula XXIV, as described hereinbelow.

A compound of Formula 104 corresponds to the structure:

wherein —A—A—, —B—B—, and R³ are as defined in Formula IV, and R¹¹ is C₁to C₄ alkyl.

A compound of Formula 104A corresponds to Formula 104 wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula 104 may be prepared by thermal decomposition of acompound of Formula 103.

A compound of Formula 103 corresponds to the structure:

wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104.

A compound of Formula 103A corresponds to Formula 103 wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula 103 may be prepared by reaction of a correspondingcompound of Formula 102 with a dialkyl malonate in the presence of abase such as an alkali metal alkoxide.

A compound of Formula 102 corresponds to the structure:

wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104.

A compound of Formula 102A corresponds to Formula 102 wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula 102 may be prepared by reaction of a correspondingcompound of Formula 101 with a trialkyl sulfonium compound in thepresence of a base.

A compound of Formula 101 corresponds to the structure:

wherein —A—A—, —B—B—, R³ and R¹¹ are as defined in Formula 104.

A compound of Formula 101A corresponds to Formula 101 wherein each of—A—A— and —B—B— is —CH₂—CH₂—, and R³ is hydrogen. Compounds within thescope of Formula 101 may be prepared by reaction of11α-hydroxyandrostene-3,17-dione or other compound of Formula XXXVI witha trialkyl orthoformate in the presence of an acid.

Based on the disclosure of specific reaction schemes as set outhereinbelow, it will be apparent which of these compounds have thegreatest utility relative to a particular reaction scheme. Use of thecompounds of this invention are useful as intermediates forepoxymexrenone and other steroids.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow sheet of a process for the bioconversion ofcanrenone or a canrenone derivative to the corresponding 11α-hydroxycompound;

FIG. 2 is a schematic flow sheet of a preferred process for thebioconversion of 11-α-hydroxylation of canrenone and canrenonederivatives;

FIG. 3 is a schematic flow sheet of a particularly preferred process forthe bioconversion of 11-α-hydroxylation of canrenone and canrenonederivatives;

FIG. 4 shows the particle size distribution for canrenone as prepared inaccordance with the process of FIG. 2; and

FIG. 5 shows the particle size distribution for canrenone as sterilizedin the transformation fermenter in accordance with the process of FIG.3.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, various novel process schemeshave been devised for the preparation of epoxymexrenone and othercompounds corresponding Formula I:

wherein:

—A—A— represents the group —CHR⁴—CHR⁵— or —CR⁴═CR⁵—

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,

R¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxyalkylradical, —B—B— represents the group —CHR⁶—CHR⁷— or an alpha- orbeta-oriented group:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy, orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁸ or R⁹ together with R⁶ or

R⁷ comprise a carbocyclic or heterocyclic ring structure fused to thepentacyclic D ring.

Unless stated otherwise, organic radicals referred to as “lower” in thepresent disclosure contain at most 7, and preferably from 1 to 4, carbonatoms.

A lower alkoxycarbonyl radical is preferably one derived from an alkylradical having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec.-butyl and tert.-butyl; especiallypreferred are methoxycarbonyl, ethoxycarbonyl and isopropoxycarbonyl. Alower alkoxy radical is preferably one derived from one of theabove-mentioned C₁-C₄ alkyl radicals, especially from a primary C₁-C₄alkyl radical; especially preferred is methoxy. A lower alkanoyl isradical is preferably one derived from a straight-chain alkyl havingfrom 1 to 7 carbon atoms; especially preferred are formyl and acetyl.

A methylene bridge in the 15,16-position is preferably β-oriented.

A preferred class of compounds that may be produced in accordance withthe methods of the invention are the 20-spiroxane compounds described inU.S. Pat. No. 4,559,332, i.e., those corresponding to Formula IA:

where:

—A—A— represents the group —CH₂—CH₂— or —CH═CH—, —B—B— represents thegroup —CH₂—CH₂— or an alpha- or beta-oriented group of Formula IIIA:

R¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonylradical,

X represents two hydrogen atoms, oxo or ═S Y¹ and Y² together representthe oxygen bridge —O—, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X represents H₂, also loweralkanoyloxy,

Preferably, 20-spiroxane compounds produced by the novel methods of theinvention are those of Formula I in which Y¹ and Y² together representthe oxygen bridge —O—.

Especially preferred compounds of the formula I are those in which Xrepresents oxo.

Of compounds of the 20-spiroxane compounds of Formula IA in which Xrepresents oxo there are most especially preferred those in which Y¹together with Y² represents the oxygen bridge —O—.

As already mentioned, 17β-hydroxy-21-carboxylic acid may also be in theform of their salts. There come into consideration especially metal andammonium salts, such as alkali metal and alkaline earth metal salts, forexample sodium, calcium, magnesium and, preferably, potassium, salts,and ammonium salts derived from ammonia or a suitable, preferablyphysiologically tolerable, organic nitrogen-containing base. As basesthere come into consideration not only amines, for example loweralkylamines (such as triethylamine), hydroxy-lower alkylamines [such as2-hydroxyethylamine, di-(2-hydroxyethyl)-amine ortri-(2-hydroxyethyl)-amine], cycloalkylamines (such asdicyclohexylamine) or benzylamines (such as benzylamine andN,N′-dibenzylethylenediamine), but also nitrogen-containing heterocycliccompounds, for example those of aromatic character (such as pyridine orquinoline) or those having an at least partially saturated heterocyclicring (such as N-ethylpiperidine, morpholine, piperazine orN,N′-dimethylpiperazine).

Also included amongst preferred compounds are alkali metal salts,especially potassium salts, of compounds of the formula IA in which R¹represents alkoxycarbonyl, with X representing oxo and each of Y¹ and Y²representing hydroxy.

Especially preferred compounds of the formula I and IA are, for example,the following:

9α,11α-epoxy-7α-methoxycarbonyl-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-7α-ethoxycarbonyl-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-7α-isopropoxycarbonyl-20-spirox-4-ene-3,21-dione,

and the 1,2-dehydro analogue of each of the compounds,

9α,11α-epoxy-6α,7α-methylene-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-6α,7β-methylene-20-spirox-4-ene-3,21-dione,

9α,11α-epoxy-6α,7β;15β,16β-bismethylene-20-spirox-4-ene-3,21-dione,

and the 1,2-dehydro analogue of each of these compounds,

9α,11α-epoxy-7α-methoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-7α-ethoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-7α-isopropoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6α,7α-methylene-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6β,7β-methylene-3-oxo-pregn-4-ene-21-carboxylicacid,

9α,11α-epoxy-17β-hydroxy-6β,7β;15β,16β-bismethylene-3-oxo-pregn-4-ene-21-carboxylicacid, and alkali metal salts, especially the potassium salt or ammoniumof each of these acids, and also a corresponding 1,2-dehydro analogue ofeach of the mentioned carboxylic acids or of a salt thereof.

9α,11α-epoxy-15β, 16β-methylene-3,21-dioxo-20-spirox-4-ene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

9α,11α-epoxy-1565β,16β-methylene-3,21-dioxo-20-spiroxa-1,4-diene-7α-carboxylicacid methyl ester,

ethyl ester and isopropyl ester,

and also 99α,11α-epoxy-3-oxo-20-spirox-4-ene-7α-carboxylic acid methylester, ethyl ester and isopropyl ester,

9α,11α-epoxy-6β,6β-methylene-20-spirox-4-en-3-one,

9α,11α-epoxy-6β,7β;15β,16β-bismethylene-20-spirox-4-en-3-one,

and also9α,11α-epoxy,17β-hydroxy-17α(3-hydroxy-propyl)-3-oxo-androst-4-ene-7α-carboxylicacid methyl ester, ethyl ester and isopropyl ester,

9α,11α-epoxy,17β-hydroxy-17α-(3-hydroxypropyl)-6α,7α-methylene-androst-4-en-3-one,

9α,11α-epoxy-17β-hydroxy-17α-(3-hydroxypropyl)-6β,7β-methylene-androst-4-en-3-one,

9α,11α-epoxy-17β-hydroxy-17α-(3-hydroxypropyl)-6β,7β;15β,16β-bismethylene-androst-4-en-3-one,

including 17α-(3-acetoxypropyl) and 17α-(3-fromyloxypropyl) analogues ofthe mentioned androstane compounds,

and also 1,2-dehydro analogues of all the mentioned compounds of theandrost-4-en-3-one and 20-spirox-4-en-3-one series.

The chemical names of the compounds of the Formulae I and IA, and ofanalogue compounds having the same characteristic structural features,are derived according to current nomenclature in the following manner:for compounds in which Y¹ together with Y² represents —O—, from20-spiroxane (for example a compound of the formula IA in which Xrepresents oxo and Y¹ together with Y² represents —O— is derived from20-spiroxan-21-one); for those in which each of Y¹ and Y² representshydroxy and X represents oxo, from17β-hydroxy-17α-pregnene-21-carboxylic acid; and for those in which eachof Y¹ and Y² represents hydroxy and X represents two hydrogen atoms,from 17β-hydroxy-17α-(3-hydroxypropyl)-androstane. Since the cyclic andopen-chain forms, that is to say lactones and 17β-hydroxy-21-carboxylicacids and their salts, respectively, are so closely related to eachother that the latter may be considered merely as a hydrated form of theformer, there is to be understood hereinbefore and hereinafter, unlessspecifically stated otherwise, both in end products of the formula I andin starting materials and intermediates of analogous structure, in eachcase all the mentioned forms together.

In accordance with the invention, several separate process schemes havebeen devised for the preparation of compounds of Formula I in high yieldand at reasonable cost. Each of the synthesis schemes proceeds throughthe preparation of a series of intermediates. A number of theseintermediates are novel compounds, and the methods of preparation ofthese intermediates are novel processes.

SCHEME 1 Starting with Canrenone or Related Material

One preferred process scheme for the preparation of compounds of FormulaI advantageously begins with canrenone or a related starting materialcorresponding to Formula XIII

wherein

—A—A— represents the group —CHR⁴ —CHR⁵— or CR⁴═CR⁵—

R³, R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl,alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy,

—B—B— represents the group —CHR⁶—CHR⁷— or an alpha- or beta-orientedgroup:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy,and

R⁸ and R⁹ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, aryloxy orR⁸ and R⁹ together comprise a carbocyclic or heterocyclic ringstructure, or R⁵ and R⁹ together with R¹ or R⁷ comprise a carbocyclic orheterocyclic ring structure fused to the pentacyclic D ring. Using abioconversion process of the type illustrated in FIGS. 1 and 2, an11-hydroxy group of α-orientation is introduced in the compound ofFormula XIII, thereby producing a compound of Formula VIII:

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined above. Preferably, thecompound of Formula XIII has the structure

 and the 11α-hydroxy product has the structure

 in each of which

—A—A— represents the group —CH₂—CH₂— or —CH═CH—,

—B—B— represents the group —CH₂—CH₂— or an alpha- or beta-orientedgroup:

X represents two hydrogen atoms, oxo or ═S,

Y¹ and Y² together represent the oxygen bridge —O—, or

Y¹ represents hydroxy, and

Y² represents hydroxy, lower alkoxy or, if X

represents H₂, also lower alkanoyloxy, and salts of compounds in which Xrepresents oxo and Y² represents hydroxy-, and the compound of FormulaVIII produced in the reaction corresponds to Formula VIIIA

 wherein —A—A—, —B—B—, Y¹, Y², and X are as defined in Formula XXXA.More preferably, R⁸ and R⁹ together form the 20-spiroxane structure:

—A—A— and —B—B— are each —CH₂—CH₂—, and R³ is hydrogen.

Among the preferred organisms that can be used in this hydroxylationstep are Aspergillus ochraceus NRRL 405, Aspergillus ochraceus ATCC18500, Aspergillus niger ATCC 16888 and ATCC 26693, Aspergillus nidulansATCC 11267, Rhizopus oryzae ATCC 11145, Rhizopus stolonifer ATCC 6227b,Streptomyces fradiae ATCC 10745, Bacillus megaterium ATCC 14945,Pseudomonas cruciviae ATCC 13262, and Trichothecium roseum ATCC 12543.Other preferred organisms include Fusarium oxysporum f.sp.cepae ATCC11171 and Rhizopus arrhizus ATCC 11145.

Other organisms that have exhibited activity for this reaction includeAbsidia coerula ATCC 6647, Absidia glauca ATCC 22752, Actinomucorelegans ATCC 6476, Aspercillus flavipes ATCC 1030, Aspercillus fumigatusATCC 26:934, Beauveria bassiana ATCC 7159 and ATCC 13144,Botryosbphaeria obtusa IMI 038560, Calonectria decora ATCC 14767,Chaetomium cochliodes ATCC 10195, Corynespora cassiicola ATCC 16718,Cunninghamella blakesleeana ATCC 8688a, Cunninghamella echinulata ATCC3655, Cunninghamella elegans ATCC 9245, Curvularia clavata ATCC 22921,Curvularia lunata ACTT 12071, Cylindrocarpon radicicola ATCC 1011,Epicoccum humicola ATCC 12722, Gongronella butleri ATCC 22822, Hypomyceschrysospermus, Mortierella isabellina ATCC 42613, Mucor mucedo ATCC4605, Mucor griseo-cyanus ATCC 1207A, Myrothecium verrucaria ATCC 9095,Nocardia corallina, Paecilomyces carneus ATCC 46579, Penicillum patulumATCC 24550, Pithomyces atro-olivaceus IFO 6651, Pithomyces cynodontisATCC 26150, Pycnosporium sp. ATCC 12231, Saccharopolyspora erythrae ATCC11635, Sepedonium chrysospermum ATCC 13378, Stachylidium bicolor ATCC12672, Streptomyces hygroscopicus ATCC 27438, Streptomyces purpurascensATCC 25489, Syncephalastrum racemosum ATCC 18192, Thamnostylum piriformeATCC 8992, Thielavia terricola ATCC 13807, and Verticillium theobromaeATCC 12474.

Additional organisms that may be expected to show activity for the11α-hydroxylation include Cephalosporium aphidicola (Phytochemistry(1996), 42(2), 411-415), Cochliobolus lunatas (J. Biotechnol. (1995),42(2), 145-150), Tieghemella orchidis (Khim.-Farm.Zh. (1986), 20(7),871-876), Tieghemella hyalospora (Khim.-Farm.Zh. (1986), 20(7),871-876), Monosporium olivaceum (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Aspergillus ustus (Acta Microbiol. Pol., Ser. B. (1973),5(2), 103-110), Fusarium graminearum (Acta Microbiol. Pol., Ser. B.(1973), 5(2), 103-110), Verticillium glaucum (Acta Microbiol. Pol., Ser.B. (1973), 5(2), 103-110), and Rhizopus nigricans (J. Steroid Biochem.(1987). 28(2), 197-201).

Preparatory to production scale fermentation for hydroxylation ofcanrenone or other substrates of Formula XIII, an inoculum of cells isprepared in a seed fermentation system comprising a seed fermenter, or aseries of two or more seed fermenters. A working stock spore suspensionis introduced into the first seed fermenter, together with a nutrientsolution for growth of cells. If the volume of inoculum desired orneeded for production exceeds that produced in the first seed fermenter,the inoculum volume may be progressively and geometrically amplified byprogression through the remaining fermenters in the seed fermentationtrain. Preferably, the inoculum produced in the seed fermentation systemis of sufficient volume and viable cells for achieving rapid initiationof reaction in the production fermenter, relatively short productionbatch cycles, and high production fermenter activity. Whatever thenumber of vessels in a train of seed fermenters, the second andsubsequent seed fermenters are preferably sized so that the extent ofdilution at each step in the train is essentially the same. The initialdilution of inoculum in each seed fermenter can be approximately thesame as the dilution in the production fermenter. Canrenone or otherFormula XIII substrate is charged to the production fermenter along withinoculum and nutrient solution, and the hydroxylation reaction conductedthere.

The spore suspension charged to the seed fermentation system is from avial of working stock spore suspension taken from a plurality of vialsconstituting a working stock cell bank that is stored under cryogenicconditions prior to use. The working stock cell bank is in turn derivedfrom a master stock cell bank that has been prepared in the followingmanner. A spore specimen obtained from an appropriate source, e.g.,ATCC, is initially suspended in an aqueous medium such as, for example,saline solution, nutrient solution or a surfactant solution, (e.g., anonionic surfactant such as Tween 20 at a concentration of about 0.001%by weight), and the suspension distributed among culture plates, eachplate bearing a solid nutrient mixture, typically based on anon-digestible polysaccharide such as agar, where the spores arepropagated. The solid nutrient mixture preferably contains between about0.5% and about 5% by weight glucose, between about 0.05% and about 5% byweight of a nitrogen source, e.g., peptone, between about 0.05% andabout 0.5% by weight of a phosphorus source, e.g., an ammonium or alkalimetal phosphate such as dipotassium hydrogen phosphate, between about0.25% and about 2.5% by weight yeast lysate or extract (or other aminoacid source such as meat extract or brain heart infusion), between about1% and about 2% by weight agar or other non-digestible polysaccharide.Optionally, the solid nutrient mixture may further comprise and/orcontain between about 0.1% and about 5% by weight malt extract. The pHof the solid nutrient mixture is preferably between about 5.0 and about7.0, adjusted as required by alkali metal hydroxide or orthophosphoricacid. Among useful solid growth media are the following:

1. Solid Medium #1: 1% glucose, 0.25% yeast extract, 0.3% K₂HPO₄ and 2%agar (Bacto); pH adjusted to 6.5 with 20% NaOH.

2. Solid Medium #2: 2% peptone (Bacto), 1% yeast extract (Bacto), 2%glucose and 2% agar (Bacto); pH adjusted to 5 with 10% H₃PO₄.

3. Solid Medium #3: 0.1% peptone (Bacto), 2% malt extract (Bacto), 2%glucose and 2% agar (Bacto); pH as is 5.3.

4. Liquid Medium: 5% blackstrap molasses, 0.5% cornsteep liquor, 0.25%glucose, 0.25% NaCl and 0.5% KH₂PO₄, pH adjusted to 5.8.

5. Difco Mycological agar (low pH).

The number of agar plates used in the development of a master stock cellbank can be selected with a view to future demands for master stock, buttypically about 15 to about 30 plates are so prepared. After a suitableperiod of growth, e.g., 7 to 10 days, the plates are scraped in thepresence of an aqueous vehicle, typically saline or buffer, forharvesting the spores, and the resulting master stock suspension isdivided among small vials, e.g., one ml. in each of a plurality of 1.5ml vials. To prepare a working stock spore suspension for use inresearch or production fermentation operations, the contents of one ormore of these second generation master stock vials can be distributedamong and incubated on agar plates in the manner described above for thepreparation of master stock spore suspension. Where routinemanufacturing operations are contemplated, as many as 100 to 400 platesmay be used to generate second generation working stock. Each plate isscraped into a separate working stock vial, each vial typicallycontaining one ml of the inoculum produced. For permanent preservation,both the master stock suspension and the second generation productioninoculum are advantageously stored in the vapor space of a cryogenicstorage vessel containing liquid N₂ or other cryogenic liquid.

In the process illustrated in FIG. 1, aqueous growth medium is preparedwhich includes a nitrogen source such as peptone, a yeast derivative orequivalent, glucose, and a source of phosphorus such as a phosphatesalt. Spores of the microorganism are cultured in this medium in theseed fermentation system. The preferred microorganism is Aspergillusochraceus NRRL 405 (ATCC 18500). The seed stock so produced is thenintroduced into the production fermenter together with the substrate ofFormula XIII. The fermentation broth is agitated and aerated for a timesufficient for the reaction to proceed to the desired degree ofcompletion.

The medium for the seed fermenter preferably comprises an aqueousmixture which contains: between about 0.5% and about 5% by weightglucose, between about 0.05% and about 5% by weight of a nitrogensource, e.g., peptone, between about 0.05% and about 0.5% by weight of aphosphorus source, e.g., an ammonium or alkali metal phosphate such asammonium phosphate monobasic or dipotassium hydrogen phosphate, betweenabout 0.25% and about 2.5% by weight yeast lysate or extract (or otheramino acid source such as distiller's solubles), between about 1% andabout 2% by weight agar or other non-digestible polysaccharide. Aparticularly preferred seed growth medium contains about 0.05% and about5% by weight of a nitrogen source such as peptone, between about 0.25%and about 2.5% by weight of autolyzed yeast or yeast extract, betweenabout 0.5% and about 5% by weight glucose, and between about 0.05% byweight and about 0.5% by weight of a phosphorus source such as ammoniumphosphate monobasic. Especially economical process operations areafforded by the use of another preferred seed culture which containsbetween about 0.5% and about 5% by weight corn steep liquor, betweenabout 0.25% and about 2.5% autolyzed yeast or yeast extract, betweenabout 0.5% and about 5% by weight glucose and about 0.05% and about 0.5%by weight ammonium phosphate monobasic. Corn steep liquor is aparticularly economical source of proteins, peptides, carbohydrates,organic acids, vitamins, metal ions, trace matters and phosphates. Mashliquors from other grains may be used in place of, or in addition to,corn steep liquor. The pH of the medium is preferably adjusted withinthe range of between about 5.0 and about 7.0, e.g., by addition of analkali metal hydroxide or orthophosphoric acid. Where corn steep liquorserves as the source of nitrogen and carbon, the pH is preferablyadjusted within the range of. about 6.2 to about 6.8. The mediumcomprising peptone and glucose is preferably adjusted to a pH betweenabout 5.4 and about 6.2. Among useful growth media for use in seedfermentation:

1. Medium #1: 2% peptone, 2% yeast autolised (or yeast extract) and 2%glucose; pH adjusted to 5.8 with 20% NaOH.

2. Medium #2: 3% corn steep liquor, 1.5% yeast extract 0.3% ammoniumphosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20% NaOH.

Spores of the microorganism are introduced into this medium from a vialtypically containing in the neighborhood of 10⁹ spores per ml. ofsuspension. Optimal productivity of seed generation is realized wheredilution with growth medium at the beginning of a seed culture does notreduce the spore population density below about 10⁷ per ml. Preferably,the spores are cultured in the seed fermentation system until the packedmycelial volume (PMV) in the seed fermenter is at least about 20%,preferably 35% to 45%. Since the cycle in the seed fermentation vessel(or any vessel of a plurality which comprise a seed fermentation train)depends on the initial concentration in that vessel, it may be desirableis to provide two or three seed fermentation stages to accelerate theoverall process. However, it is preferable to avoid the use ofsignificantly more than three seed fermenters in series, since activitymay be compromised if seed fermentation is carried through an excessivenumber of stages. The seed culture fermentation is conducted underagitation at a temperature in the range of about 23° to about 37° C.,preferably in range of between about 24° and about 28° C.

Culture from the seed fermentation system is introduced into aproduction fermenter, together with a production growth medium. In oneembodiment of the invention, non-sterile canrenone or other substrate ofFormula XIII serves as the substrate for the reaction. Preferably, thesubstrate is added to the production fermenter in the form of a 10% to30% by weight slurry in growth medium. To increase the surface areaavailable for 11α-hydroxylation reaction, the particle size of theFormula XIII substrate is reduced by passing the substrate through anoff line micronizer prior to introduction into the fermenter. A sterilenutrient feed stock containing glucose, and a second sterile nutrientsolution containing a yeast derivative such as autolyzed yeast (orequivalent amino acid formulation based on alternative sources such asdistiller's solubles), are also separately introduced. The mediumcomprises an aqueous mixture containing: between about 0.5% and about 5%by weight glucose, between about 0.05% and about 5% by weight of anitrogen source, e.g., peptone, between about 0.05% and about 0.5% byweight of a phosphorus source, e.g., an ammonium or alkali metalphosphate such as dipotassium hydrogen phosphate, between about 0.25%and about 2.5% by weight yeast lysate or extract (or other amino acidsource such as distiller's solubles), between about 1% and about 2% byweight agar or other non-digestible polysaccharide. A particularlypreferred production growth medium contains about 0.05% and about 5% byweight of a nitrogen source such as peptone, between about 0.25% andabout 2.5% by weight of autolyzed yeast or yeast extract, between about0.5% and about 5% by weight glucose, and between about 0.05% and about0.5% by weight of a phosphorus source such as ammonium phosphatemonobasic. Another preferred production medium contains between about0.5% and about 5% by weight corn steep liquor, between about 0.25% andabout 2.5% autolyzed yeast or yeast extract, between about 0.5% andabout 5% by weight glucose and about 0.05% and about 0.5% by weightammonium phosphate monobasic. The pH of the production fermentationmedium is preferably adjusted in the manner described above for the seedfermentation medium, with the same preferred ranges for the pH ofpeptone/glucose based media and corn steep liquor based media,respectively. Useful bioconversion growth media are set forth below:

1. Medium #1: 2% peptone, 2% yeast autolised (or yeast extract) and 2%glucose; pH adjusted to 5.8 with 20% NaOH.

2. Medium #2: 1% peptone, 1% yeast autolised (or yeast extract) and 2%glucose; pH adjusted to 5.8 with 20% NaOH.

3. Medium #3: 0.5% peptone, 0.5% yeast autolised (or yeast extract) and0.5% glucose; pH adjusted to 5.8 with 20% NaOH.

4. Medium #4: 3% corn steep liquor, 1.5% yeast extract 0.3% ammoniumphosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20% NaOH.

5. Medium #5: 2.55% corn steep liquor, 1.275% yeast extract 0.255%ammonium phosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20%NaOH.

6. Medium #6: 2.1% corn steep liquor, 1.05% yeast extract 0.21% ammoniumphosphate monobasic and 3% glucose; pH adjusted to 6.5 with 20% NaOH.

Non-sterile canrenone and sterile nutrient solutions are chain fed tothe production fermenter in five to twenty, preferably ten to fifteen,preferably substantially equal, portions each over the production batchcycle. Advantageously, the substrate is initially introduced in anamount sufficient to establish a concentration of between about 0.1% byweight and about 3% by weight, preferably between about 0.5% and about2% by weight, before inoculation with seed fermentation broth, thenadded periodically, conveniently every 8 to 24 hours, to a cumulativeproportion of between about 1% and about 8% by weight. Where additionalsubstrate is added every 8 hour shift, total addition may be slightlylower, e.g., 0.25% to 2.5% by weight, than in the case where substrateis added only on a daily basis. In the latter instance cumulativecanrenone addition may need to be in the range 2% to about 8% by weight.The supplemental nutrient mixture fed during the fermentation reactionis preferably a concentrate, for example, a mixture containing betweenabout 40% and about 60% by weight sterile glucose, and between about 16%and about 32% by weight sterile yeast extract or other sterile source ofyeast derivative (or other amino acid source). Since the substrate fedto the production fermenter of FIG. 1 is non-sterile, antibiotics areperiodically added to the fermentation broth to control the growth ofundesired organisms. Antibiotics such as kanamycin, tetracycline, andcefalexin can be added without disadvantageously affecting growth andbioconversion. Preferably, these are introduced into the fermentationbroth in a concentration, e.g., of between about 0.0004% and about0.002% based on the total amount of the broth, comprising, e.g., betweenabout 0.0002% and about 0.0006% kanamicyn sulfate, between about 0.0002%and about 0.006% tetracycline HCl and/or between about 0.001% and about0.003% cefalexin, again based on the total amount of broth.

Typically, the production fermentation batch cycle is in theneighborhood of 80-160 hours. Thus, portions of each of the Formula XIIIsubstrate and nutrient solutions are typically added every 2 to 10hours, preferably every 4 to 6 hours. Advantageously, an antifoam isalso incorporated in the seed fermentation system, and in the productionfermenter.

Preferably, in the process of FIG. 1, the inoculum charge to theproduction fermenter is about 0.5 to about 7%, more preferably about 1to about 2%, by volume based on the total mixture in the fermenter, andthe glucose concentration is maintained between about 0.01% and about1.0%, preferably between about 0.025% and about 0.5%, more preferablybetween about 0.05% and about 0.25% by weight with periodic additionsthat are preferably in portions of about 0.05% to about 0.25% by weight,based on the total batch charge. The fermentation temperature isconveniently controlled within a range of about 200 to about 37° C.,preferably about 24° C. to about 28° C., but it may be desirable to stepdown the temperature during the reaction, e.g., in 20° C. increments, tomaintain the packed mycelium volume (PMV) below about 60%, morepreferably below about 50%, and thereby prevent the viscosity of thefermentation broth from interfering with satisfactory mixing. If thebiomass growth extends above the liquid surface, substrate retainedwithin the biomass may be carried out of the reaction zone and becomeunavailable for the hydroxylation reaction. For productivity, it isdesirable to reach a PMV in the range of 30 to 50%, preferably 35% to45%, within the first 24 hours of the fermentation reaction, butthereafter conditions are preferably managed to control further growthwithin the limits stated above. During reaction, the pH of thefermentation medium is controlled at between about 5.0 and about 6.5,preferably between about 5.2 and about 5.8, and the fermenter isagitated at a rate of between about 400 and about 800 rpm. A dissolvedoxygen level of at least about 10% of saturation is achieved by aeratingthe batch at between about 0.2 and about 1.0 vvm, and maintaining thepressure in the head space of the fermenter at between about atmosphericand about 1.0 bar gauge, most preferably in the neighborhood of about0.7 bar gauge. Agitation rate may also been increased as necessary tomaintain minimum dissolved oxygen levels. Advantageously, the dissolvedoxygen is maintained at well above 10%, in fact as high as 50% topromote conversion of substrate. Maintaining the pH in the range of5.5±0.2 is also optimal for bioconversion. Foaming is controlled asnecessary by addition of a common antifoaming agent. After all substratehas been added, reaction is preferably continued until the molar ratioof Formula VIII product to remaining unreacted Formula XIII substrate isat least about 9 to 1. Such conversion may be achieve within the 80-160hour batch cycle indicated above.

It has been found that high conversions are associated with depletion ofinitial nutrient levels below the initial charge level, and bycontrolling aeration rate and agitation rate to avoid splashing ofsubstrate out of the liquid broth. In the process of FIG. 1, thenutrient level was depleted to and then maintained at no greater thanabout 60%, preferably about 50%, of the initial charge level; while inthe processes of FIGS. 2 and 3, the nutrient level was reduced to andmaintained at no greater than about 80%, preferably about 70%, of theinitial charge level. Aeration rate is preferably no greater than onevvm, more preferably in the range of about 0.5 vvm; while agitation rateis preferably not greater than 600 rpm.

A particularly preferred process for preparation of a compound ofFormula VIII is illustrated in FIG. 2. Again the preferred microorganismis Aspergillus ochraceus NRRL 405 (ATCC 18500). In this process, growthmedium preferably comprises between about 0.5% and about 5% by weightcorn steep liquor, between about 0.5% and about 5% by weight glucose,between about 0.1% and about 3% by weight yeast extract, and betweenabout 0.05% and about 0.5% by weight ammonium phosphate. However, otherproduction growth media as described herein may also be used. The seedculture is prepared essentially in the manner described for the processof FIG. 1, using any of the seed fermentation media described herein. Asuspension of non-micronized canrenone or other Formula XIII substratein the growth medium is prepared aseptically in a blender, preferably ata relatively high concentration of between about 10% and about 30% byweight substrate. Preferably, aseptic preparation may comprisesterilization or pasteurization of the suspension after mixing. Theentire amount of sterile substrate suspension required for a productionbatch is introduced into the production fermenter at the beginning ofthe batch, or by periodical chain feeding. The particle size of thesubstrate is reduced by wet milling in an on-line shear pump whichtransfers the slurry to the production fermenter, thus obviating theneed for use of an off line micronizer. Where aseptic conditions areachieved by pasteurization rather than sterilization, the extent ofagglomeration may be insignificant, but the use of a shear pump may bedesirable to provide positive control of particle size. Sterile growthmedium and glucose solution are introduced into the production fermenteressentially in the same manner as described above. All feed componentsto the production fermenter are sterilized before introduction, so thatno antibiotics are required.

Preferably, in operation of the process of FIG. 2, the inoculum isintroduced into the production fermenter in a proportion of betweenabout 0.5% and about 7%, the fermentation temperature is between about20° and about 37° C., preferably between about 24° C. and about 28° C.,and the pH is controlled between about 4.4 and about 6.5, preferablybetween about 5.3 and about 5.5, e.g., by introduction of gaseousammonia, aqueous ammonium hydroxide, aqueous alkali metal hydroxide, ororthophosphoric acid. As in the process of FIG. 1, the temperature ispreferably trimmed to control growth of the biomass so that PMV does notexceed 55-60%. The initial glucose charge is preferably between about 1%and about 4% by weight, most preferably 2.5% to 3.5% by weight, but ispreferably allowed to drift below about 1.0% by weight duringfermentation. Supplemental glucose is fed periodically in portions ofbetween about 0.2% and about 1.0% by weight based on the total batchcharge, so as to maintain the glucose concentration in the fermentationzone within a range of between about 0.1% and about 1.5% by weight,preferably between about 0.25% and about 0.5% by weight. Optionally,nitrogen and phosphorus sources may be supplemented along with glucose.However, because the entire canrenone charge is made at the beginning ofthe batch cycle, the requisite supply of nitrogen and phosphorus bearingnutrients can also be introduced at that time, allowing the use of onlya glucose solution for supplementation during the reaction. The rate andnature of agitation is a significant variable. Moderately vigorousagitation promotes mass transfer between the solid substrate and theaqueous phase. However, a low shear impeller should be used to preventdegradation of the myelin of the microorganisms. Optimal agitationvelocity varies within the range of 200 to 800 rpm, depending on culturebroth viscosity, oxygen concentration, and mixing conditions as affectedby vessel, baffle and impeller configuration. Ordinarily, a preferredagitation rate is in the range of 350-600 rpm. Preferably the agitationimpeller provides a downward axially pumping function so as to assist ingood mixing of the fermented biomass. The batch is preferably aerated ata rate of between about 0.3 and about 1.0 vvm, preferably 0.4 to 0.8vvm, and the pressure in the head space of the fermenter is preferablybetween about 0.5 and about 1.0 bar gauge. Temperature, agitation,aeration and back pressure are preferably controlled to maintaindissolved oxygen in the range of at least about 10% by volume during thebioconversion. Total batch cycle is typically between about 100 andabout 140 hours.

Although the principle of operation for the process of FIG. 2 is basedon early introduction of substantially the entire canrenone charge, itwill be understood that growth of the fermentation broth may be carriedout before the bulk of the canrenone is charged. Optionally, someportion of the canrenone can also be added later in the batch.Generally, however, at least about 75% of the sterile canrenone chargeshould be introduced into the transformation fermenter within 48 hoursafter initiation of fermentation. Moreover, it is desirable to introduceat least about 25% by weight canrenone at the beginning of thefermentation, or at least within the first 24 hours in order to promotegeneration of the bioconversion enzyme(s).

In a further preferred process as illustrated in FIG. 3, the entirebatch charge and nutrient solution are sterilized in the productionfermentation vessel prior to the introduction of inoculum. The nutrientsolutions that may be used, as well as the preferences among them, areessentially the same as in the process of FIG. 2. In this embodiment ofthe invention, the shearing action of the agitator impeller breaks downthe substrate agglomerates that otherwise tend to form uponsterilization. It has been found that the reaction proceedssatisfactorily if the mean particle size of the canrenone is less thanabout 200μ and at least 75% by weight of the particles are smaller than240μ. The use of a suitable impeller, e.g., a disk turbine impeller, atan adequate velocity in the range of 200 to 800 rpm, with a tip speed ofat least about 400 cm/sec., has been found to provide a shear ratesufficient to maintain such particle size characteristics despite theagglomeration that tends to occur upon sterilization within theproduction fermenter. The remaining operation of the process of FIG. 3is essentially the same as the process of FIG. 2. The processes of FIGS.2 and 3 offer several distinct advantages over the process of FIG. 1. Aparticular advantage is the amenability to use of a low cost nutrientbase such as corn steep liquor. But further advantages are realized ineliminating the need of antibiotics, simplifying feeding procedures, andallowing for batch sterilization of canrenone or other Formula XIIIsubstrate. Another particular advantage is the ability to use a simpleglucose solution rather than a complex nutrient solution forsupplementation during the reaction cycle.

In processes depicted in FIGS. 1 to 3, the is product of Fig. VIII is acrystalline solid which, together with the biomass, may be separatedfrom the reaction broth by filtration or low speed centrifugation.Alternatively, the product can be extracted from the entire reactionbroth with organic solvents. Product of Formula VIII is recovered bysolvent extraction. For maximum recovery, both the liquid phase filtrateand the biomass filter or centrifuge cake are treated with extractionsolvent, but usually ≧95% of the product is associated with the biomass.Typically, hydrocarbon, ester, chlorinated hydrocarbon, and ketonesolvents may be used for extraction. A preferred solvent is a ethylacetate. Other typically suitable solvents include toluene and methylisobutyl ketone. For extraction from the liquid phase, it may beconvenient to use a volume of solvent approximately equal to the volumeof reaction solution which it contacts. To recover product the from thebiomass, the latter is suspended in the solvent, preferably in largeexcess relative to the initial charge of substrate, e.g., 50 to 100 ml.solvent per gram of initial canrenone charge, and the resultingsuspension preferably refluxed for a period of 20 minutes to severalhours to assure transfer of product to the solvent phase from recessesand pores of the biomass. Thereafter, the biomass is removed byfiltration or centrifugation, and the filter cake preferably washed withboth fresh solvent and deionized water. Aqueous and solvent washes arethen combined and the phases allowed to separate. Formula VIII productis recovered by crystallization from the solution. To maximize yield,the mycelium is contacted twice with fresh solvent. After settling toallow complete separation of the aqueous phase, product is recoveredfrom the solvent phase. Most preferably, the solvent is removed undervacuum until crystallization begins, then the concentrated extract iscooled to a temperature of 0° to 20° C., preferably about 100 to about15° C. for a time sufficient for crystal precipitation and growth,typically 8 to 12 hours.

The processes of FIG. 2, and especially that of FIG. 3, are particularlypreferred. These processes operate at low viscosity, and are amenable toclose control of process parameters such as pH, temperature anddissolved oxygen. Moreover, sterile conditions are readily preservedwithout resort to antibiotics.

The bioconversion process is exothermic, so that heat should be removed,using a jacketed fermenter or a cooling coil within the productionfermenter. Alternatively, the reaction broth may be circulated throughan external heat exchanger. Dissolved oxygen is preferably maintained ata level of at least about 5%, preferably at least about 10%, by volume,sufficient to provide energy for the reaction and assure conversion ofthe glucose to CO₂ and H₂O, by regulating the rate of air introducedinto the reactor in response to measurement of oxygen potential in thebroth. The pH is preferably controlled at between about 4.5 and about6.5.

In each of the alternative processes for 11-hydroxylation of thesubstrate of Formula XIII, productivity is limited by mass transfer fromthe solid substrate to the aqueous phase, or the phase interface, wherereaction is understood to occur. As indicated above, productivity is notsignificantly limited by mass transfer rates so long as the particlemean particle size of the substrate is reduced to less than about 300μ,and at least 75% by weight of the particles are smaller than 240μ.However, productivity of these processes may be further enhanced incertain alternative embodiments which provide a substantial charge ofcanrenone or other Formula XIII substrate to the production fermenter inan organic solvent. According to one option, the substrate is dissolvedin a water-immiscible solvent and mixed with the aqueous growth mediuminoculum and a surfactant. Useful water-immiscible solvents inlcude, forexample, DMF, DMSO, C₆-C₁₂ fatty acids, C₆-C₁₂ n-alkanes, vegetableoils, sorbitans, and aqueous surfactant solutions. Agitation of thischarge generates an emulsion reaction system having an extendedinterfacial area for mass transfer of substrate from the organic liquidphase to the reaction sites.

A second option is to initially dissolve the substrate in a watermiscible solvent such as acetone, methylethyl ketone, methanol, ethanol,or glycerol in a concentration substantially greater than its solubilityin water. By preparing the initial substrate solution at elevatedtemperature, solubility is increased, thereby further increasing theamount of solution form substrate introduced into the reactor andultimately enhancing the reactor payload. The warm substrate solution ischarged to the production fermentation reactor along with the relativelycool aqueous charge comprising growth medium and inoculum. When thesubstrate solution is mixed with the aqueous medium, precipitation ofthe substrate occurs. However, under conditions of substantialsupersaturation and moderately vigorous agitation, nucleation is favoredover crystal growth, and very fine particles of high surface area areformed. The high surface area promotes mass transfer between the liquidphase and the solid substrate. Moreover, the equilibrium concentrationof substrate in the aqueous liquid phase is also enhanced in thepresence of a water-miscible solvent. Accordingly, productivity ispromoted.

Although the microorganism may not necessarily tolerate a highconcentration of organic solvent in the aqueous phase, a concentrationof ethanol, e.g., in the range of about 3% to about 5% by weight, can beused to advantage.

A third option is to solubilize the substrate in an aqueous cyclodextrinsolution. Illustrative cyclodextrins includehydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin. The molar ratioof substrate:cyclodextrin can be about 1:1 to about 1:1.5,substrate:cyclodextrin. The substrate:cyclodextrin mixture can then beadded aseptically to the bioconversion reactor.

11α-Hydroxycanrenone and other products of the 11α-hydroxylation process(Formulae VIII and VIIIA) are novel compounds, which may be isolated byfiltering the reaction medium, and extracting the product from thebiomass collected on the filtration medium. Conventional organicsolvents, e.g., ethyl acetate, acetone, toluene, chlorinatedhydrocarbons, and methyl isobutyl ketone may be used for the extraction.The product of Formula VIII may then be recrystallized from an organicsolvent of the same type. The compounds of Formula VIII have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA.

Preferably, the compounds of Formula VIII correspond to Formula VIIIA inwhich —A—A— and —B—B— are —CH₂—CH₂—, R³ is hydrogen, lower alkyl orlower alkoxy, and R⁸ and R⁹ together constitute the 20-spiroxane ring:

Further in accordance with the process of scheme 1, the compound ofFormula VIII is reacted under alkaline conditions with a source ofcyanide ion to produce an enamine compound of Formula VII

wherein —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined above. Where thesubstrate corresponds to Formula VIIIA, the product is of Formula VIIA

wherein —A—A—, —B—B—, R³, Y¹, Y², and X are as defined in Formula XIII.

Cyanidation of the 11α-hydroxyl substrate of Formula VIII may be carriedout by reacting it with a cyanide ion source such as a ketonecyanohydrin, most preferably acetone cyanohydrin, in the presence of abase and a alkali metal salt, most preferably LiCl. Alternatively,cyanidation can be effected without a cyanohydrin by using an alkalimetal cyanide in the presence of an acid.

In the ketone cyanohydrin process, the reaction is conducted insolution, preferably using an aprotic polar solvent such asdimethylformamide or dimethyl sulfoxide. Formation of the enaminerequires at least two moles of cyanide ion source per mole of substrate,and preferably a slight excess of the cyanide source is used. The baseis preferably a nitrogenous base such as a dialkylamine, trialkylamine,alkanolamine, pyridine or the like. However, inorganic bases such asalkali metal carbonates or alkali metal hydroxides can also be used.Preferably, the substrate of Formula VIII is initially present in aproportion of between about 20 and about 50% by weight and the base ispresent in a proportion of between 0.5 to two equivalents per equivalentof substrate. The temperature of the reaction is not critical, butproductivity is enhanced by operation at elevated temperature. Thus, forexample, where triethylamine is used as the base, the reaction isadvantageously conducted in the range of about 80° C. to about 90° C. Atsuch temperatures, the reaction proceeds to completion in about 5 toabout 20 hours. When diisopropylethyl amine is used as the base and thereaction is conducted at 105° C., the reaction is completed at 8 hours.At the end of the reaction period, the solvent is removed under vacuumand the residual oil dissolved in water and neutralized to pH 7 withdilute acid, preferably hydrochloric. The product precipitates from thissolution, and is thereafter washed with distilled water and air dried.Liberated HCN may be stripped with an inert gas and quenched in analkaline solution. The dried precipitate is taken up in chloroform orother suitable solvent, then extracted with concentrated acid, e.g., 6NHCl. The extract is neutralized to pH 7 by addition of an inorganicbase, preferably an alkali metal hydroxide, and cooled to a temperaturein the range of 0° C. The resulting precipitate is washed and dried,then recrystallized from a suitable solvent, e.g., acetone, to produce aproduct of Formula VII suitable for use in the next step of the process.

Alternatively, the reaction may be conducted in a aqueous solvent systemcomprising water-miscible organic solvent such as methanol or in abiphasic system comprising water and an organic solvent such as ethylacetate. In this alternative, product may be recovered by diluting thereaction solution with water, and thereafter extracting the productusing an organic solvent such as methylene chloride or chloroform, andthen back extracting from the organic extract using concentrated mineralacid, e.g., 2N HCl. See U.S. Pat. No. 3,200,113.

According to a still further alternative, the reaction may be conductedin a water-miscible solvent such as dimethylformamide,dimethylacetamide, N-methyl, pyrolidone or dimethyl sulfoxide, afterwhich the reaction product solution is diluted with water and renderedalkaline, e.g., by addition of an alkali metal carbonate, then cooled to0° to 10° C., thereby causing t he product to precipitate. Preferably,the system is quenched with an alkali metal hypohalite or other reagenteffective to prevent evolution of cyanide. After filtration and washingwith water, the precipitated product is suitable for use in the nextstep of the process.

According to a still further alternative, the enamine product of FormulaVII may be produced by reaction of a substrate of Formula VIII in thepresence of a proton source, with an excess of alkali metal cyanide,preferably NaCN, in an aqueous solvent comprising an aproticwater-miscible polar solvent such as dimethylformamide ordimethylacetamide. The proton source is preferably a mineral acid or C₁to C₅ carboxylic acid, sulfuric acid being particularly preferred.Anomalously, no discrete proton source need be added where thecyanidation reagent is commercial LiCN in DMF.

Cyanide ion is preferably charged to the reactor in a proportion ofbetween about 2.05 and about 5 molar equivalents per equivalent ofsubstrate. The mineral acid or other proton source is believed topromote addition of HCN across the 4,5 and 6,7 double bonds, and ispreferably present in a proportion of at least one mole equivalent permole equivalent substrate; but the reaction system should remain basicby maintaining an excess of alkali metal cyanide over acid present.Reaction is preferably carried out at a temperature of at least about75° C., typically 60° C. to 100° C., for a period of about 1 to about 8hours, preferably about 1.5 to about 3 hours. At the end of the reactionperiod, the reaction mixture is cooled, preferably to about roomtemperature; and the product enamine is precipitated by acidifying thereaction mixture and mixing it with cold water, preferably at about icebath temperature. Acidification is believed to close the 17-lactone,which tends to open under the basic conditions prevailing in thecyanidation. The reaction mixture is conveniently acidified using thesame acid that is present during the reaction, preferably sulfuric acid.Water is preferably added in a proportion of between about 10 and about50 mole equivalents per mole of product.

The compounds of Formula VII are novel compounds and have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula VIIcorrespond to Formula VIIA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

Most preferably the compound of Formula VII is5′R(5′α),7′β-20′-Aminohexadecahydro-11β-hydroxy-10′α,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile.

In the next step of the Scheme 1 synthesis, the enamine of Formula VIIis hydrolyzed to produce a diketone compound of Formula VI

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII. Anyaqueous organic or mineral acid can be used for the hydrolysis.Hydrochloric acid is preferred. To enhance productivity, awater-miscible organic solvent, such as a lower alkanol, is preferablyused as a cosolvent. The acid should be present in proportion of atleast one equivalent per equivalent of Formula VII substrate. In anaqueous system, the enamine substrate VII can be substantially convertedto the diketone of Formula VII in a period of about 5 hours at about 80°C. Operation at elevated temperature increases productivity, buttemperature is not critical. Suitable temperatures are selected based onthe volatility of the solvent system and acid.

Preferably, the enamine substrate of Formula VII corresponds to FormulaVIIA

and the diketone product corresponds to Formula VIA

in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined in FormulaVIIIA.

At the end of the reaction period, the solution is cooled to 0° and 25°C. to crystallize the product. The product crystals may berecrystallized from a suitable solvent such as isopropanol or methanolto produce a product of Formula VI suitable for use in the next step ofthe process; but recrystallization is usually not necessary. Theproducts of Formula VI are novel compounds which have substantial valueas intermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Preferably, the compounds of Formula VIcorrespond to Formula VIA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

Most preferably, the compound of Formula VI is4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2,H)-carbonitrile.

In a particularly preferred embodiment of the invention, the productenamine of Formula VII is produced from the compound of Formula VIII inthe manner described above, and converted in situ to the diketone ofFormula VI. In this embodiment of the invention, a formula VIIIsubstrate is reacted with an excess of alkali metal cyanide in anaqueous solvent containing a proton source, or optionally an excess ofketone cyanohydrin in the presence of a base and LiCl, as describedhereinabove. However, instead of cooling the reaction mixture,acidifying, and adding water in proportions calculated to causeprecipitation of the enamine, substantial cooling of the reactionmixture is preferably avoided. Water and an acid, preferably a mineralacid such as sulfuric, are indeed added to mixture at the end of thecyanidation reaction, and the proportion of acid added is sufficient toneutralize excess alkali metal cyanide, which ordinarily requiresintroduction of at least one molar equivalent acid per mole of FormulaVIII substrate, preferably between about 2 and about 5 mole equivalentsper equivalent substrate. However, the temperature is maintained at highenough, and the dilution greater enough, so that substantialprecipitation is avoided and hydrolysis of the enamine to the diketoneis allowed to proceed in the liquid phase. Thus, the process proceedswith minimum interruption and high productivity. Hydrolysis ispreferably conducted at a temperature of at least 80° C., morepreferably in the range of about 90° C. to about 100° C., for a periodof typically about 1 to about 10 hours, more preferably about 2 to about5 hours. Then the reaction mixture is cooled, preferably to atemperature of between about 0° C. and about 15° C., advantageously inan ice bath to about 5° C. to about 10° C., for precipitation of theproduct diketone of Formula VI The solid product may be recovered, as byfiltration, and impurities removed by washing with water.

In the next step of the Scheme 1 synthesis, the diketone compound ofFormula VI is reacted with a metal alkoxide to open up the ketone bridgebetween the 4 and 7 positions, cleave the bond between the carbonylgroup and the 4-carbon, and form an α-oriented alkanoyloxycarbonylsubstituent at the 7 position and eliminating cyanide at the 5-carbon.The product of this reaction is a hydroxyester compound corresponding toFormula V

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII, and R¹is lower alkoxycarbonyl or hydroxycarbonyl. The metal alkoxide used inthe reaction corresponds to the formula R¹⁰OM where M is alkali metaland R¹⁰ corresponds to the alkoxy substituent of R¹. Yields of thisreaction are most satisfactory when the metal alkoxide is K or Namethoxide, but other lower alkoxides can be used. A K alkoxide isparticularly preferred. Phenoxides, other aryloxides may also be used,as well as arylsulfides. The reaction is conveniently carried out in thepresence of an alcohol corresponding to the formula R¹⁰OH where R¹⁰ isas defined above. Other conventional solvents may be used. Preferably,the Formula VI substrate is present in a proportion of between about 2%and about 12% by weight, more preferably at least about 6% by weight andR¹⁰OM is present in a proportion of between about 0.5 and about 4 molesper mole of substrate. Temperature is not critical but elevatedtemperature enhances productivity. Reaction time is typically betweenabout 4 and about 24 hours, preferably about 4 to 16 hours.Conveniently, the reaction is carried out at atmospheric refluxtemperature depending on the solvent used.

In the conversion of the diketone of Formula VI to the hydroxyester ofFormula VI, by-product cyanide ion can react with the product to form5-cyanoester. Because the equilibrium is more favorable at lowconcentrations, the reaction is preferably run at rather high dilution,e.g., as high as 40:1 for reaction with Na methoxide. It has been foundthat significantly higher productivity can be realized by use of Kmethoxide rather than Na methoxide, because a dilution in the range ofabout 20:1 is generally sufficient to minimize the extent of reversecyanidation where K methoxide is the reagent.

In accordance with the invention, it has been further discovered thatthe reverse cyanidation reaction may be inhibited by taking appropriatechemical or physical measures to remove by-product cyanide ion from thereaction zone. Thus, in a further embodiment of the invention, thereaction of the diketone with alkali metal alkoxide may be carried outin the presence of an precipitating agent for cyanide ion such as, forexample, a salt, comprising a cation which forms an insoluble cyanidecompound. Such salts may, for example, include zinc iodide, ferricsulfate, or essentially any halide, sulfate or other salt of an alkalineearth or transition metal that is more soluble than the correspondingcyanide. If zinc iodide is present in proportions in the range of aboutone equivalent per equivalent diketone substrate, it has been observedthat the productivity of the reaction is increased substantially ascompared to the process as conducted in the absence of an alkali metalhalide.

Even where a precipitating agent is used for removal of cyanide ion, itremains preferable to run at fairly high dilution, but by use of aprecipitating agent the solvent to diketone substrate molar ratio may bereduced significantly compared to reactions in the absence of suchagent. Recovery of the hydroxyester of Formula V can be carried outaccording to either the extractive or non-extractive proceduresdescribed below.

Preferably, the diketone substrate of Formula VI corresponds to FormulaVIA

and the hydroxyester product corresponds to Formula VA

in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined in FormulaXIII and R¹ is as defined in Formula V.

The products of Formula V are novel compounds which have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula Vcorrespond to Formula VA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

Most preferably, the compound of Formula V is Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

The compound of Formula V may be isolated by acidifying the reactionsolution, e.g., with concentrated HCl, cooling to ambient temperature,and extracting the product with an organic solvent such as methylenechloride or ethyl acetate. The extract is washed with an aqueousalkaline wash solution, dried and filtered, after which the solvent isremoved. Alternatively, the reaction solution containing the product ofFormula V may be quenched with-concentrated acid. The product solutionis concentrated, cooled to 0° to 25° C. and the product solid isisolated by filtration.

According to a preferred mode of recovery of the product of Formula V,methanol and HCN are removed by distillation after the conclusion of thereaction period, with water and acid being added before or during thedistillation. Addition of water before the distillation simplifiesoperations, but progressive addition during the distillation allows thevolume in the still to be maintained substantially constant. Product ofFormula V crystallizes from the still bottoms as the distillationproceeds. This mode of recovery provides a high quality crystallineproduct without extraction operations.

In accordance with a further alternative, the reaction solutioncontaining the product of Formula V may be quenched with mineral acid,e.g., 4N HCl, after which the solvent is removed by distillation.Removal of the solvent is also effective for removing residual HCN fromthe reaction product. It has been found that multiple solventextractions for purification of the compound of Formula V are notnecessary where the compound of Formula V serves as an intermediate in aprocess for the preparation of epoxymexrenone, as described herein. Infact, such extractions can often be entirely eliminated. Where solventextraction is used for product purification, it is desirable tosupplement the solvent washes with brine and caustic washes. But wherethe solvent extractions are eliminated, the brine and caustic washes aretoo. Eliminating the extractions and washes significantly enhances theproductivity of the process, without sacrificing yield or productquality, and also eliminates the need for drying of the washed solutionwith a dessicant such as sodium sulfate. The crude11α-hydroxy-7α-alkanoyloxycarbonyl product is taken up again in thesolvent for the next reaction step of the process, which is theconversion of the 11-hydroxy group to a good leaving group at the 11position thereby producing a compound of Formula IV:

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII, R¹ isas defined in Formula V, and R² is lower arylsulfonyloxy,alkylsulfonyloxy, acyloxy or halide. Preferably, the 11α-hydroxyl isesterified by reaction with a lower alkylsulfonyl halide, an acyl halideor an acid anhydride which is added to the solution containing theintermediate product of Formula V. Lower alkylsulfonyl halides, andespecially methanesulfonyl chloride, are preferred. Alternatively, the11-α hydroxy group could be converted to a halide by reaction of asuitable reagent such as thionyl bromide, thionyl chloride, sulfurylchloride or oxalyl chloride. Other reagents for forming 11α-sulfonicacid esters include tosyl chloride, benzenesulfonyl chloride andtrifluoromethanesulfonic anhydride. The reaction is conducted in asolvent containing a hydrogen halide scavenger such as triethylamine orpyridine. Inorganic bases such as K or Na carbonate can also be used.The initial concentration of the hydroxyester of Formula V is preferablybetween about 5% and about 50% by weight. The esterification reagent ispreferably present in slight excess. Methylene chloride is aparticularly suitable solvent for the reaction, but other solvents suchas dichloroethane, pyridine, chloroform, methyl ethyl ketone,dimethoxyethane, methyl isobutyl ketone, acetone, other ketones, ethers,acetonitrile, toluene, and tetrahydrofuran can also be employed. Thereaction temperature is governed primarily by the volatility of thesolvent. In methylene chloride, the reaction temperature is preferablyin the range of between about −10° C. and about 10° C.

Preferably, the hydroxyester substrate of Formula V corresponds toFormula VA

and the product corresponds to Formula IVA

in each of which —A—A—, —B—B—, Y¹, Y² and X are as defined in FormulaXIII, R¹ is lower alkanoyloxycarbonyl or hydroxycarbonyl, and R² is asdefined in Formula IV.

The products of Formula IV are novel compounds which have substantialvalue as intermediates for the preparation of compounds of Formula I,and especially of Formula IA. Preferably, the compounds of Formula IVcorrespond to Formula VA in which —A—A— and —B—B— are —CH₂—CH₂—, R³ ishydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹ together constitutethe 20-spiroxane ring:

Most preferably, the compound of Formula IV is Methyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone.

If desired, the compound of Formula IV may be isolated by removal of thesolvent. Preferably, the reaction solution is first washed with anaqueous alkaline wash solution, e.g., 0.5-2N NaoH, followed by an acidwash, e.g., 0.5-2N HCl. After removal of the reaction solvent, theproduct is recrystallized, e.g., by taking the product up in methylenechloride and then adding another solvent such as ethyl ether whichlowers the solubility of the product of Formula IV, causing it toprecipitate in crystalline form.

In the recovery of the product of Formula IV, or in preparation of thereaction solution for conversion of the Formula IV intermediate to theintermediate of Formula II as is further described hereinbelow, allextractions and/or washing steps may be dispensed with if the solutionis instead treated with ion exchange resins for removal of acidic andbasic impurities. The solution is treated first with an anion exchangeresin, then with a cation exchange resin. Alternatively, the reactionsolution may first be treated with inorganic adsorbents such as basicalumina or basic silica, followed by a dilute acid wash. Basic silica orbasic alumina may typically be mixed with the reaction solution in aproportion of between about 5 and about 50 g per kg of product,preferably between about 15 and about 20 g per kg product. Whether ionexchange resins or inorganic adsorbents are used, the treatment can becarried out by simply slurrying the resin or inorganic adsorbent withthe reaction solution under agitation at ambient temperature, thenremoving the resin or inorganic adsorbent by filtration.

In an alternative and preferred embodiment of the invention, the productcompound of Formula IV is recovered in crude form as a concentratedsolution by removal of a portion of the solvent. This concentratedsolution is used directly in the following step of the process, which isremoval of the 11α-leaving group from the compound of Formula IV,thereby producing an enester of Formula II:

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII, and R¹is as defined in Formula V. For purposes of this reaction, the R²substituent of the compound of Formula IV may be any leaving group theabstraction of which is effective for generating a double bond betweenthe 9- and 11-carbons. Preferably, the leaving group is a loweralkylsulfonyloxy or acyloxy substituent which is removed by reactionwith an acid and an alkali metal salt. Mineral acids can be used, butlower alkanoic acids are preferred. Advantageously, the reagent for thereaction further includes an alkali metal salt of the alkanoic acidutilized. It is particularly preferred that the leaving group comprisemesyloxy and the reagent for the reaction comprise formic acid or aceticacid and an alkali metal salt of one of these acids or another loweralkanoic acid. Where the leaving group is mesyloxy and the removalreagent is formic acid and potassium formate a relatively high ratio of9,11 to 11,12-olefin is observed. If free water is present duringremoval of the leaving group, impurities tend to be formed, particularlya 7,9-lactone

which is difficult to remove from the final product. Hence, aceticanhydride or other drying agent is used to remove the water present informic acid. The free water content of the reaction mixture beforereaction should be maintained at a level below about 0.5%, preferablybelow about 0.1% by weight, as measured by Karl Fischer analysis forwater, based on total reaction solution. Although it is preferred thatthe reaction mixture be kept as dry as practicable, satisfactory resultshave been realized with 0.3% by weight water. Preferably, the reactioncharge mixture contains between about 4% and about 50% by weight of thesubstrate of Formula IV in the alkanoic acid. Between about 4% and about20% by weight of the alkali metal salt of the acid is preferablyincluded. Where acetic anhydride is used as the drying agent, it ispreferably present in a proportion of between about 0.05 moles and about0.2 moles per mole of alkanoic acid.

It has been found that proportions of by-product 7,9-lactone and11,12-olefin in the reaction mixture is relatively low where theelimination reagent comprises a combination of trifluoroacetic acid,trifluoroacetic anhydride and potassium acetate as the reagent forelimination of the leaving group and formation of the enester(9,11-olefin). Trifluoroacetic anhydride serves as the drying agent, andshould be present in a proportion of at least about 3% by weight, morepreferably at least about 15% by weight, most preferably about 20% byweight, based on the trifluoroacetic acid eliminating reagent.

Alternatively, the 11α-leaving groups from the compound of Formula IV,may be eliminated to produce an enester of Formula II by heating asolution of Formula IV in an organic solvent such as DMSO, DMF or DMA.

Further in accordance with the invention, the compound of Formula IV isreacted initially with an alkenyl alkanoate such as isopropenyl acetatein the presence of an acid such as toluene sulfonic acid or an anhydrousmineral acid such as sulfuric acid to form the 3-enol ester:

of the compound of Formula IV. Alternatively, the 3 enol ester can beformed by treatment with an acid anhydrides and base such as acetic acidand sodium acetate. Further alternatives include treatment with ketenein the presence of an acid to produce IV(Z). The intermediate of FormulaIV(Z) is thereafter reacted with an alkali metal formate or acetate inthe presence of formic or acetic acid to produce the Δ-9,11 enol acetateof Formula IV(Y):

which can then be converted to the enester of Formula II in an organicsolvent, preferably an alcohol such as methanol, by either thermaldecomposition of the enol acetate or reaction thereof with an alkalimetal alkoxide. The elimination reaction is highly selective to theenester of Formula II in preference to the 11,12-olefin and 7,9-lactone,and this selectivity is preserved through conversion of the enol acetateto the enone.

Preferably, the substrate of Formula IV corresponds to Formula IVA

and the enester product corresponds to Formula IIA

in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined in FormulaXIII and R¹ is as defined in Formula V.

If desired, the compound of Formula II may be isolated by removing thesolvent, taking up the solid product in cold water, and extracting withan organic solvent, such as ethyl acetate. After appropriate washing anddrying steps, the product is recovered by removing the extractionsolvent. The enester is then dissolved in a solvent appropriate for theconversion to the product of Formula I. Alternatively, the enester canbe isolated by adding water to the concentrated product solution andfiltering the solid product, thereby preferentially removing the7,9-lactone. Conversion of the substrate of Formula II to the product ofFormula IA may be conducted in the manner described in U.S. Pat. No.4,559,332 which is expressly incorporated herein by reference, or morepreferably by the novel reaction using a haloacetamide promoter asdescribed below.

In another embodiment of the invention, the hydroxyester of Formula Vmay be converted to the enester of Formula II without isolation of theintermediate compound of Formula IV. In this method, the hydroxyester istaken up in a an organic solvent, such as methylene chloride; and eitheran acylating agent, e.g., methanesulfonyl chloride, or halogenatingreagent, e.g., sulfuryl chloride, is added to the solution. The mixtureis agitated and, where halogenation is involved, an HCl scavenger suchas imidazole is added. Mixing of base with the solution is highlyexothermic, and should therefore be conducted at a controlled rate withfull cooling. After the base addition, the resulting mixture is warmedto moderate temperature, e.g., 0° C. to room temperature or slightlyabove, and reacted for a period of typically 1 to 4 hours. Afterreaction is complete, the solvent is stripped, preferably under highvacuum (e.g., 24″ to 28″ Hg) conditions at −100 to +15° C., morepreferably about 0° to about 5° C., to concentrate the solution andremove excess base. The substrate is then redissolved in an organicsolvent, preferably a halogenated solvent such as methylene chloride forconversion to the enester.

The leaving group elimination reagent is preferably prepared by mixingan organic acid, an organic acid salt and a drying agent, preferablyformic acid, alkali metal formate and acetic anhydride, respectively, ina dry reactor. Addition of acetic anhydride is exothermic and results inrelease of CO, so the addition rate must be controlled accordingly. Topromote the removal of water, the temperature of this reaction ispreferably maintained in the range of 60° to 90° C., most preferablyabout 65° to about 75° C. This reagent is then added to the productsolution of the compound of Formula IV to effect the eliminationreaction. After 4-8 hours, the reaction mixture is preferably heated toa temperature of at least about 85° C., but not above about 95° C. untilall volatile distillate has been removed, and then for an additionalperiod to complete the reaction, typically about 1 to 4 hours. Thereaction mixture is cooled, and after recovery by standard extractiontechniques, the enester may be recovered as desired by evaporating thesolvent.

It has further been found that the enester of Formula II may berecovered from the reaction solution by an alternative procedure whichavoids the need for extraction steps following the elimination reaction,thereby providing savings in cost, improvement in yield and/orimprovement in productivity. In this process, the enester product isprecipitated by dilution of the reaction mixture with water afterremoval of formic acid. The product is then isolated by filtration. Noextractions are required.

According to a further alternative for conversion of the hydroxyester ofFormula V to the enester of Formula II without isolation of the compoundof Formula IV, the 11α-hydroxy group of the Formula V hydroxyester isreplaced by halogen, and the Formula II enester is then formed in situby thermal dehydro halogenation. Replacement of the hydroxy group byhalogen is effected by reaction with sulfuryl halide, preferablysulfuryl chloride, in the cold in the presence of a hydrogen halidescavenger such as imidazole. The hydroxyester is dissolved in a solventsuch as tetrahydrofuran and cooled to 0° C. to −70° C. The sulfurylhalide is added and the reaction mixture is warmed to moderatetemperature, e.g., room temperature, for a time sufficient to completethe elimination reaction, typically 1 to 4 hours. The process of thisembodiment not only combines two steps into one, but eliminates the useof: a halogenated reaction solvent; an acid (such as acetic); and adrying reagent (acetic anhydride or sodium sulfate). Moreover, thereaction does not require refluxing conditions, and avoids thegeneration of by-product CO which results when acetic acid is used as adrying reagent.

In accordance with a particularly preferred embodiment of the invention,the diketone compound of Formula VI can be converted to epoxymexrenoneor other compound of Formula I without isolating any intermediate inpurified form. In accordance with this preferred process, the reactionsolution containing the hydroxyester is quenched with a strong acidsolution, cooled to ambient temperature and then extracted with anappropriate extraction solvent. Advantageously, an aqueous solution ofinorganic salt, e.g., 10% by weight saline solution, is added to thereaction mixture prior to the extraction. The extract is washed anddried by azeotropic distillation for removal of the methanol solventremaining from the ketone cleavage reaction.

The resulting concentrated solution containing between about 5% andabout 50% by weight compound of Formula V is then contacted in the coldwith an acylating or alkylsulfonylating reagent to form the sulfonicester or dicarboxylic acid ester. After the alkylsulfonation orcarboxylation reaction is complete the reaction solution is passed overan acidic and then a basic exchange resin column for the removal ofbasic and acidic impurities. After each pass, the column is washed withan appropriate solvent, e.g., methylene chloride, for the recovery ofresidual sulfonic or dicarboxylic ester therefrom. The combined eluateand wash fractions are combined and reduced, preferably under vacuum, toproduce a concentrated solution containing the sulfonic ester ordicarboxylic ester of Formula IV. This concentrated solution is thencontacted with a dry reagent comprising an agent effect for removal ofthe 11α-ester leaving group and abstraction of hydrogen to form a 9,11double bond. Preferably, the reagent for removal of the leaving groupcomprises the formic acid/alkali metal formate/acetic anhydride dryreagent solution described above. After reaction is complete, thereaction mixture is cooled and formic acid and/or other volatilecomponents are removed under vacuum. The residue is cooled to ambienttemperature, subjected to appropriate washing steps, and then dried togive a concentrated solution containing the enester of Formula II. Thisenester may then be converted to epoxymexrenone or other compound ofFormula I using the method described herein, or in U.S. Pat. No.4,559,332.

In an especially preferred embodiment of the invention, the solvent isremoved from the reaction solution under vacuum, and the product ofFormula IV is partitioned between water and an appropriate organicsolvent, e.g., ethyl acetate. The aqueous layer is then back extractedwith the organic solvent, and the back extract washed with an alkalinesolution, preferably a solution of an alkali metal hydroxide containingan alkali metal halide. The organic phase is concentrated, preferablyunder vacuum, to yield the enester product of Formula II. The product ofFormula II may then be taken up in an organic solvent, e.g., methylenechloride, and further reacted in the manner described in the '332 patentto produce the product of Formula I.

Where trihaloacetonitrile is used in the epoxidation reaction, it hasbeen found that the selection of solvent is important, with halogenatedsolvents being highly preferred, and methylene chloride being especiallypreferred. Solvents such as dichloroethane and chlorobenzene givereasonably satisfactory yields, but yields are generally better in amethylene chloride reaction medium. Solvents such as acetonitrile andethyl acetate generally give poor yields, while reaction in solventssuch as methanol or water/tetrahydrofuran give little of the desiredproduct.

Further in accordance with the present invention, it has been discoveredthat numerous improvements in the synthesis of epoxymexrenone can berealized by use of a trihaloacetamide rather than a trihaloacetonitrileas a peroxide activator for the epoxidation reaction. In accordance witha particularly preferred process, the epoxidation is carried out byreaction of the substrate of Formula IIA with hydrogen peroxide in thepresence of trichloroacetamide and an appropriate buffer. Preferably,the reaction is conducted in a pH in the range of about 3 to about 7,most preferably between about 5 and about 7. However, despite theseconsiderations, successful reaction has been realized outside thepreferred pH ranges.

Especially favorable results are obtained with a buffer comprisingdipotassium hydrogen phosphate, and/or with a buffer comprising acombination of dipotassium hydrogenphosphate and potassium dihydrogenphosphate in relative proportions of between about 1:4 and about 2:1,most preferably in the range of about 2:3. Borate buffers can also beused, but generally give slower conversions than dipotassium phosphateor K₂HPO₄, or K₂HPO₄/KH₂PO₄ mixtures. Whatever the makeup of the buffer,it should provide a pH in the range indicated above. Aside from theoverall composition of the buffer or the precise pH it may impart, ithas been observed that the reaction proceeds much more effectively if atleast a portion of the buffer is comprised of dibasic hydrogenphosphateion. It is believed that this ion may participate essentially as ahomogeneous catalyst in the formation of an adduct or complex comprisingthe promoter and hydroperoxide ion, the generation of which may in turnbe essential to the overall epoxidation reaction mechanism. Thus, thequantitative requirement for dibasic hydrogenphosphate (preferably fromK₂HPO₄) may be only a small catalytic concentration. Generally, it ispreferred that HPO₄ be present in a proportion of at least about 0.1equivalents, e.g., between about 0.1 and about 0.3 equivalents, perequivalent substrate.

The reaction is carried out in a suitable solvent, preferably methylenechloride, but alternatively other halogenated solvents such aschlorobenzene or dichloroethane can be used. Toluene and mixtures oftoluene and acetonitrile have also been found satisfactory. Withoutcommitting to a particular theory, it is posited that the reactionproceeds most effectively in a two phase system in which a hydroperoxideintermediate is formed and distributes to the organic phase of low watercontent, and reacts with the substrate in the organic phase. Thus thepreferred solvents are those in which water solubility is low. Effectiverecovery from toluene is promoted by inclusion of another solvent suchas acetonitrile.

In the conversion of substrates of Formula II to products of Formula I,toluene provides a process advantage since the substrates are freelysoluble in toluene and the products are not. Thus, the productprecipitates during the reaction when conversions reach the 40-50%range, producing a three phase mixture from which the product can beconveniently separated by filtration. Methanol, ethyl acetate,acetonitrile alone, THF and THF/water have not proved as to be aseffective as the halogenated solvents or toluene in carrying out theconversion of this step of the process.

While trichloroacetamide is a highly preferred reagent, othertrihaloacetamides such as trifluoroacetamide can also be used.Trihalomethylbenzamide, and other compounds having an arylene moietybetween the electron withdrawing trihalomethyl group and the carbonyl ofthe amide, may also be useful. 3,3,3-Trihalopropionamides may also beused, but with less favorable results. Generically, the peroxideactivator may correspond to the formula:

R⁰C(O)NH₂

where R⁰ is a group having an electron withdrawing strength (as measuredby sigma constant) at least as high as that of the monochloromethylgroup. More particularly, the peroxide activator may correspond to theformula:

where X¹, X², and X³ are independently selected from among halo,hydrogen, alkyl, haloalkyl and cyano and cyanoalkyl, and R^(P) isselected from among arylene and —(CX⁴X⁵)_(n)—, where n is 0 or 1, atleast one of X¹, X², X³, X⁴ and X⁵ being halo or perhaloalkyl. Where anyof X¹, X², X³, X⁴ or X⁵ is not halo, it is preferably haloalkyl, mostpreferably perhaloalkyl. Particularly preferred activators include thosein which n is 0 and at least two of X¹, X² and X³ are halo; or in whichall of X¹, X², X³, X⁴ and X⁵ are halo or perhaloalkyl. Each of X¹, X²X³, X⁴ and X⁵ is preferably Cl or F, most preferably Cl, though mixedhalides may also be suitable, as may perchloralkyl or perbromoalkyl andcombinations thereof.

Preferably, the peroxide activator is present in a proportion of atleast about 1 equivalents, more preferably between about 1.5 and about 2equivalents, per equivalent of substrate initially present. Hydrogenperoxide should be charged to the reaction in at least modest excess, oradded progressively as the epoxidation reaction proceeds. Although thereaction consumes only one to two equivalents of hydrogen peroxide permole of substrate, hydrogen peroxide is preferably charged insubstantial excess relative to substrate and activator initiallypresent. Without limiting the invention to a particular theory, it isbelieved that the reaction mechanism involves formation of an adduct ofthe activator and OOH⁻, that the formation of this reaction isreversible with the equilibrium favoring the reverse reaction, and thata substantial initial excess of hydrogen peroxide is therefore necessaryin order to drive the reaction in the forward direction. Temperature ofthe reaction is not narrowly critical, and may be effectively carriedout within the range of 0° to 100° C. The optimum temperature depends onthe selection of solvent. Generally, the preferred temperature isbetween about 20° C. and 30° C., but in certain solvents, e.g., toluenethe reaction may be advantageously conducted in the range of 60° -70° C.At 25° C., reaction typically requires less than 10 hours, typically 3to 6 hours. If needed additional activator and hydrogen peroxide may beadded at the end of the reaction cycle to achieve complete conversion ofthe substrate.

At the end of the reaction cycle, the aqueous phase is removed, theorganic reaction solution is preferably washed for removal of watersoluble impurities, after which the product may be recovered by removalof the solvent. Before removal of solvent, the reaction solution shouldbe washed, at with least a mild to moderately alkaline wash, e.g.,sodium carbonate. Preferably, the reaction mixture is washedsuccessively with: a mild reducing solution such as a weak (e.g. 3% byweight) solution of sodium sulfite in water; an alkaline solution, e.g.,NaOH or KOH (preferably about 0.5N); an acid solution such as HCl(preferably about 1N); and a final neutral wash comprising water orbrine, preferably saturated brine to minimize product losses. Prior toremoval of the reaction solvent, another solvent such as an organicsolvent, preferably ethanol may be advantageously added, so that theproduct may be recovered by crystallization after distillation forremoval of the more volatile reaction solvent.

It should be understood that the novel epoxidation method utilizingtrichloroacetamide or other novel peroxide activator has applicationwell beyond the various schemes for the preparation of epoxymexrenone,and in fact may be used for the formation of epoxides across olefinicdouble bonds in a wide variety of substrates subject to reaction in theliquid phase. The reaction is particularly effective in unsaturatedcompounds in which the olefinic carbons are tetrasubstituted andtrisubstituted, i.e., R^(a)R^(b)C═CR^(c)R^(d) and R^(a)R^(b)C═CR^(c)RHwhere R^(a) to R^(d) represent substituents other than hydrogen. Thereaction proceeds most rapidly and completely where the substrate is acyclic compound with a trisubstituted double, or either a cyclic oracyclic compound with tetrasubstituted double bonds. Exemplarysubstrates for this reaction include Δ-9,11-canrenone, and

Because the reaction proceeds more rapidly and completely withtrisubstituted and tetrasubstituted double bonds, it is especiallyeffective for selective epoxidation across such double bonds incompounds that may include other double bonds where the olefinic carbonsare monosubstituted, or even disubstituted.

It should be further understood that the reaction may be used toadvantage in the epoxidation of monosubstituted or even disubstituteddouble bonds, such as the 11,12-olefin in various steroid substrates.However, because it preferentially epoxidizes the more highlysubstituted double bonds, e.g., the 9,11-olefin, with high selectivity,the process of this invention is especially effective for achieving highyields and productivity in the epoxidation steps of the various reactionschemes described elsewhere herein.

The improved process has been shown to be particularly advantageousapplication to the preparation of:

by epoxidation of:

Multiple advantages have been demonstrated for the process of theinvention in which trichloroacetamide is used in place oftrihaloacetonitrile as the oxygen transfer reagent for the epoxidationreaction. The trichloroacetamide reagent system provides tightregiocontrol for epoxidation across trisubstituted double withdisubstituted and α,β-keto olefins in the same molecular structure.Thus, reaction yield, product profile and final purity are substantiallyenhanced. It has further been discovered that the substantial excessoxygen generation observed with the use of trihaloacetonitrile is notexperienced with trichloroacetamide, imparting improved safety to theepoxidation process. Further in contrast to the trichloroacetonitrilepromoted reaction, the trichloroacetamide reaction exhibits minimumexothermic effects, thus facilitating control of the thermal profile ofthe reaction. Agitation effects are observed to be minimal and reactorperformance more consistent, a further advantage over thetrichloroacetonitrile process. The reaction is more amenable to scaleupthan the trichloroacetonitrile promoted reaction. Product isolation andpurification is simple, there is no observable Bayer-Villager oxidationof carbonyl function (peroxide promoted conversion of ketone to ester)as experienced, e.g., using m-chloroperoxybenzoic acid or other peracidsand the reagent is inexpensive, readily available, and easily handled.

The novel epoxidation method of the invention is highly useful as theconcluding step of the synthesis of Scheme 1. In a particularlypreferred embodiment, the overall process of Scheme 1 proceeds asfollows:

The second of novel reaction schemes (Scheme 2) of this invention startswith canrenone or other substrate corresponding to Formula XIII

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII. In thefirst step of this process, the substrate of Formula XIII is convertedto a product of Formula XII

using a cyanidation reaction scheme substantially the same as thatdescribed above for conversion of the substrate of Formula VIII to theintermediate of Formula VII. Preferably, the substrate of Formula XIIIcorresponds to Formula XIIIA

and the enamine product corresponds to Formula XIIA

in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined in FormulaXIII.

In the second step of scheme 2, the enamine of Formula XII is hydrolyzedto an intermediate diketone product of Formula XI

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII, usinga reaction scheme substantially the same as that described above forconversion of the substrate of Formula VIII to the intermediate ofFormula VII. Preferably, the substrate of Formula XII corresponds toFormula XIIA

and the diketone product corresponds to Formula XIA

in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined in FormulaVIIIA.

Further in accordance with reaction scheme 2, the diketone of Formula XIis reacted with an alkali metal alkoxide to form mexrenone or otherproduct corresponding to Formula X,

in each of which —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in FormulaVIII. R¹ is as defined in Formula V.

The process is carried out using substantially the same reaction schemethat is described above for the conversion of the compounds of FormulaVI to those of Formula V. Preferably, the substrate of Formula XIcorresponds to Formula XIA

and the intermediate product corresponds to Formula XA

in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined in FormulaXIII. R¹ is as defined in Formula V.

Canrenone and other compounds of Formula X are next 9α-hydroxylated by anovel bioconversion process to yield products of Formula IX

where —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII, and R¹is as defined in Formula V. Among the organisms that can be used in thishydroxylation step are Nocardia conicruria ATCC 31548, Nocardia aurentiaATCC 12674, Corynespora cassiicola ATCC 16718, Streptomyceshydroscovicus ATCC 27438, Mortierella isabellina ATCC 42613, Beauvriabassiana ATCC 7519, Penicillum purpurogenum ATCC 46581, Hypomyceschrysospermus IMI 109891, Thamnostylum piriforme ATCC 8992,Cunnignhamella blakesleeana ATCC 8688a, Cunningnhamella echinulata ATCC3655, Cunninghamella elegans ATCC 9245, Trichothecium roseum ATCC 12543,Epicoccum humicola ATCC 12722, Saccharopolyspora eythrae ATCC 11635,Beauvria bassiana ATCC 13144, Arthrobacter simplex, Bacteriumcyclooxydans ATCC 12673, Cylindrocarpon radicicola ATCC 11011, Nocardiaaurentia ATCC 12674, Nocardia canicruria, Norcardia restrictus ATCC14887, Pseudomonas testosteroni ATCC 11996, Rhodococcus equi ATCC 21329,Mycobacterium fortuitum ATCC-6842, and Rhodococcus rhodochrous ATCC19150. The reaction is carried out substantially in the manner describedabove in connection with FIGS. 1 and 2. The process of FIG. 1 isparticularly preferred.

Growth media useful in the bioconversions preferably contain betweenabout 0.05% and about 5% by weight available nitrogen; between about0.5% and about 5% by weight glucose; between about 0.25% and about 2.5%by weight of a yeast derivative; and between about 0.05%, and about 0.5%by weight available phosphorus. Particularly preferred growth mediainclude the following:

soybean meal: between about 0.5% and about 3% by weight glucose; betweenabout 0.1% and about 1% by weight soybean meal; between about 0.05% andabout 0.5% by weight alkali metal halide; between about 0.05% and about560.5% by weight of a yeast derivative such as autolyzed yeast or yeastextract; between about 0.05% and about 0.5% by weight of a phosphatesalt such as K₂HPO₄; pH=7;

peptone-yeast extract-glucose: between about 0.2% and about 2% by weightpeptone; between about 0.05% and about 0.5% by weight yeast extract; andbetween about 2% and about 5% by weight glucose;

Mueller-Hinton: between about 10% and about 40% by weight beef infusion;between about 0.35% and about 8.75% by weight casamino acids; betweenabout 0.15% and about 0.7% by weight starch.

Fungi can be grown in soybean meal or peptone nutrients, whileactinomycetes and eubacteria can be grown in soybean meal (plus 0.5% to1% by weight carboxylic acid salt such as Na formate forbiotransformations) or in Mueller-Hinton broth.

The production of 11β-hydroxymexrenone from mexrenone by fermentation isdiscussed in Example 19.

The products of Formula IX are novel compounds, which may be separatedby filtration, washed with a suitable organic solvent, e.g., ethylacetate, and recrystallized from the same or a similar solvent. Theyhave substantial value as intermediates for the preparation of compoundsof Formula I, and especially of Formula IA. Preferably, the compounds ofFormula IX correspond to Formula IXA in which —A—A— and —B—B— are—CH₂—CH₂—, R³ is hydrogen, lower alkyl or lower alkoxy, and R⁸ and R⁹together constitute the 20-spiroxane ring:

In the next step of synthesis scheme 2, the product of Formula IX isreacted with a dehydration reagent to produce a compound of Formula II

wherein —A—A—, R³, —B—B—, R⁸ and R⁹ are as defined in Formula VIII, andR¹ is as defined in Formula V. Where the substrate corresponds toFormula IXA, the product is of Formula IIA

in each of which —A—A—, —B—B—, Y¹, Y², and X are as defined in FormulaXIII and R¹ is as defined in Formula V.

In the final step of this synthesis scheme, the product of Formula II isconverted to that of Formula I by epoxidation in accordance with themethod described in U.S. Pat. No. 4,559,332; or preferably by the novelepoxidation method of the invention as described hereinabove.

In a particularly preferred embodiment, the overall process of Scheme 2proceeds as follows:

SCHEME 3

The synthesis in this case begins with a substrate corresponding toFormula XX

where —A—A— and R³ are as defined in Formula VIII, —B—B— is as definedin Formula VIII except that neither R⁶ nor R⁷ is part of a ring fused tothe D ring at the 16,17 positions, and R²⁶ is lower alhyl, preferablymethyl. Reaction of the substrate of Formula XX with a sulfonium ylideproduces the epoxide intermediate corresponding to Formula XIX

wherein —A—A—, R³, —B—B—, and R²⁶ are as defined in Formula XX.

In the next step of synthesis scheme 3, the intermediate of Formula XIXis converted to a further intermediate of Formula XVIII

wherein —A—A—, R³, and —B—B— are as defined in Formula XX. In this step,Formula XIX substrate is converted to Formula XVIII intermediate byreaction with NaCH(COOEt)₂ in the presence of a base in a solvent.Exposure of the compound of Formula XVIII to heat water and an alkalihalide produces a decarboxylated intermediate compound corresponding toFormula XVII

wherein —A—A—, R³, and —B—B— are as defined in Formula XX. The processfor conversion of the compound of Formula XX to the compound of FormulaXVII corresponds essentially to that described in U.S. Pat. Nos.3,897,417, 3,413,288 and 3,300,489, which are expressly incorporatedherein by reference. While the substrates differ, the reagents,mechanisms and conditions for introduction of the 17-spirolactone moietyare essentially the same.

Reaction of the intermediate of Formula XVII with a dehydrogenationreagent yields the further intermediate of Formula XVI.

where —A—A—, R³ and —B—B— are as defined above.

Typically useful dehydrogenation reagents includedichlorodicyanobenzoquinone (DDQ) and chloranil(2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, the dehydrogenationcould be achieved by a sequential halogenation at the carbon-6 followedby dehydrohalogenation reaction.

The intermediate of Formula XVI is next converted to the enamine ofFormula XV

wherein —A—A—, R³, and —B—B— are as defined in Formula XX. Conversion isby cyanidation essentially in the manner described above for theconversion of the 11α-hydroxy compound of Formula VIII to the enamine ofFormula VII. Typically, the cyanide ion source may be an alkali metalcyanide. The base is preferably pyrrolidine and/or tetramethylguanidine.A methanol solvent may be used.

The products of Formula XV are novel compounds, which may be isolated bychromatography. These and other novel compounds of Formula AXV havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Compounds of Formula AXVcorrespond to the structure

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined above. In the mostpreferred compounds of Formula XV, and —A—A— and —B—B— are —CH2—CH2—.

In accordance with the hydrolysis described above for producing thediketone compounds of Formula VI, the enamines of Formula XV may beconverted to the diketones of Formula XIV

wherein —A—A—, R³, and —B—B— are as defined in Formula XX. Particularlypreferred for the synthesis of epoxymexrenone are those compounds ofFormula XIV which also fall within the scope of Formula VIA.

The products of Formula XIV are novel compounds, which may be isolatedby precipitation. These and other novel compounds of Formula AXIV havesubstantial value as intermediates for the preparation of compounds ofFormula I, and especially of Formula IA. Compounds of Formula AXIVcorrespond to the structure

where —A—A—, —B—B—, R³, R⁸ and R⁹ are as defined above. In the mostpreferred compounds of Formula AXIV and XIV, —A—A— and —B—B— are—CH₂—CH₂—.

The compounds of Formula XIV are further converted to compounds ofFormula XXXI using essentially the process described above forconverting the diketone of Formula VI to the hydroxyester of Formula V.In this instance, it is necessary to isolate the intermediate XXXI

before further conversion to a product of Formula XXXII

wherein —A—A— and —B—B— are as defined in Formula XX. Preferredcompounds of Formula XXXI are those which fall within Formula IIA. Thecompounds of Formula XXXI are converted to compounds of Formula XXXIIusing the method described hereinabove or in U.S. Pat. No. 4,559,332. Ina particularly preferred embodiment, the overall process of Scheme 3proceeds as follows:

SCHEME 4

The first three steps of Scheme 4 are the same as those of Scheme 3,i.e., preparation of an intermediate of Formula XVII starting with acompound corresponding to Formula XX.

Thereafter, the intermediate of Formula XVII is epoxidized, for example,using the process of U.S. Pat. No. 4,559,332 to produce the compound ofFormula XXIV

wherein —A—A—, R³, and —B—B— are as defined in Formula XX. However, in aparticularly preferred embodiment of the invention, the substrate ofFormula XVII is epoxidized across the 9,11-double bond using anoxidation reagent comprising an amide type peroxide activator, mostpreferably trichloroacetamide, according to the process as describedabove in Scheme 1 for the conversion of the enester of Formula II to theproduct of Formula I. The conditions and proportions of reagents forthis reaction are substantially as described for the conversion of theFormula II enester to epoxymexrenone.

It has been found that the epoxidation of the substrate of Formula XVIIcan also be effected in very good yield using a peracid such as, forexample, m-chloroperoxybenzoic acid. However, the trichloroacetamidereagent provides superior results in minimizing the formation ofBayer-Villager oxidation by-product. The latter by-product can beremoved, but this requires trituration from a solvent such as ethylacetate, followed by crystallization from another solvent such asmethylene chloride. The epoxy compound of Formula XXIV is dehydrogenatedto produce a double bond between the 6- and 7-carbons by reaction with adehydrogenation (oxidizing) agent such as DDQ or chloranil, or using thebromination/dehydrobromination (or otherhalogenation/dehydrohalogenation) sequence, to produce another novelintermediate of Formula XXIII

wherein —A—A—, and —B—B— are as defined in Formula XX. Particularlypreferred compounds of Formula XXIII are those in which —A—A— and —B—B—are as defined in Formula XIII.

While direct oxidation is effective for the formation of the product ofFormula XXIII, the yields are generally low. Preferably, therefore, theoxidation is carried out in two steps, first halogenating the substrateof Formula XXIV at the C-6 position, then dehydrohalogenating to the6,7-olefin. Halogenation is preferably effected with an N-halo organicreagent such as, for example, N-bromosuccinamide. Bromination is carriedout in a suitable solvent such as, for example, acetonitrile, in thepresence of halogenation promoter such as benzoyl peroxide. The reactionproceeds effectively at a temperature in the range of about 500 to about100° C., conveniently at atmospheric reflux temperature in a solventsuch as carbon tetrachloride, acetonitrile or mixture thereof. However,reaction from 4 to 10 hours is typically required for completion of thereaction. The reaction solvent is stripped off, and the residue taken upa water-immiscible solvent, e.g., ethyl acetate. The resulting solutionis washed sequentially with a mild alkaline solution (such as an alkalimetal bicarbonate) and water, or preferably saturated brine to minimizeproduct losses, after which the solvent is stripped and a the residuetaken up in another solvent (such as dimethylformamide) that is suitablefor the dehydrohalogenation reaction.

A suitable dehydrohalogenation reagent, e.g.,1,4-diazabicyclo[2,2,2]octane (DABCO) is added to the solution, alongwith an alkali metal halide such as LiBr, the solution heated to asuitable reaction temperature, e.g., 60° to 80° C., and reactioncontinued for several hours, typically 4 to 15 hours, to complete thedehydrobromination. Additional dehydrobromination reagent may be addedas necessary during the reaction cycle, to drive the reaction tocompletion. The product of Formula XXIII may then be recovered, e.g., byadding water to precipitate the product which is then separated byfiltration and preferably washed with additional amounts of water. Theproduct is preferably recrystallized, for example fromdimethylformamide. The products of Formula XXIII, such as9,11-epoxycanrenone, are novel compounds, which may be isolated byextraction/crystallization. They have substantial value as intermediatesfor the preparation of compounds of Formula I, and especially of FormulaIA. For example, they may be used as substrates for the preparation ofcompounds of Formula XXII. In the most preferred compounds of FormulaXXIII, and —A—A— and —B—B— are —CH₂—CH₂—.

Using substantially the process described above for the preparation ofcompounds of Formula VII, the compounds of Formula XXIII are reactedwith cyanide ion to produce novel epoxyenamine compounds correspondingto Formula XXII

wherein —A—A—, R³, and —B—B— are as defined in Formula XX. Particularlypreferred compounds of Formula XXII are those in which —A—A— and —B—B—are as defined in Formula XIII.

The products of Formula XXII are novel compounds, which may be isolatedby precipitation and filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. In the most preferred compounds of FormulaXXII, and —A—A— and —B—B— are —CH2—CH2—.

Using substantially the process described above for preparation ofcompounds of Formula VI, the epoxyenamine compounds of Formula XXII areconverted to novel epoxydiketone compounds of Formula XXI.

The products of Formula XXI are novel compounds, which may be isolatedby precipitation and filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXI are those in which —A—A— and —B—B— are as defined in Formula XIII.In the most preferred compounds of Formula XXI, and —A—A— and —B—B— are—CH2—CH₂—.

Compounds of Formula XXI are converted to compounds of Formula XXXIIusing the epoxidation process described hereinabove or the process ofU.S. Pat. No. 4,559,332. In a particularly preferred embodiment, theoverall process of Scheme 4 proceeds as follows:

SCHEME 5

The process of scheme 5 begins with a substrate corresponding to FormulaXXIX

wherein —A—A—, and —B—B— are as defined in Formula XX. This substrate isconverted to a product of Formula XXVIII

by reaction with trimethylorthoformate. wherein —A—A—, R³, and —B—B— areas defined in Formula XX. Following the formation of Formula XXVIII, thecompounds of Formula XXIX are converted to compounds of Formula XXVIIusing the method described above for conversion of the substrate ofFormula XX to Formula XVII. Compounds of Formula XXVII have thestructure:

wherein —A—A—, and —B—B— are as defined in Formula XX, and R^(x) is anyof the common hydroxyl protecting groups.

Using the method described above for the preparation of compounds ofFormula XVI, compounds of Formula XXVII are oxidized to yield novelcompounds corresponding to Formula XXVI

wherein —A—A—, and —B—B— are as defined in Formula XX. Particularlypreferred compounds of Formulae XXIX, XXVIII, XXVII and XXVI are thosein which —A—A— and —B—B— are as defined in Formula XIII.

The products of Formula XXVI are novel compounds, which may be isolatedby precipitation/filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXVI are those in which —A—A— and —B—B— are as defined in Formula XIII.In the most preferred compounds of Formula XXVI, and —A—A— and —B—B— are—CH₂—CH₂—.

Using the method defined above for cyanidation of compounds of FormulaVIII, the novel intermediates of Formula XXVI are converted to the novel9-hydroxyenamine intermediates of Formula XXV

wherein —A—A—, R³, and —B—B— are as defined in Formula XX.

The products of Formula XXV are novel compounds, which may be isolatedby precipitation/filtration. They have substantial value asintermediates for the preparation of compounds of Formula I, andespecially of Formula IA. Particularly preferred compounds of FormulaXXV are those in which —A—A— and —B—B— are as defined in Formula XIII.In the most preferred compounds of Formula XXVI, and —A—A— and —B—B— are—CH₂—CH₂—.

Using essentially the conditions described above for the preparation ofthe diketone compounds of Formula VI, the 9-hydroxyenamine intermediatesof Formula XXV are converted to the diketone compounds of Formula XIV.Note that in this instance the reaction is effective for simultaneoushydrolysis of the enamine structure and dehydration at the 9,11positions to introduce the 9,11 double bond. The compound of Formula XIVis then converted to the compound of Formula XXXI, and thence to thecompound of Formula XIII, using the same steps that are described abovein scheme 3.

In a particularly preferred embodiment, the overall process of Scheme 5proceeds as follows:

Scheme 6 provides an advantageous method for the preparation ofepoxymexrenone and other compounds corresponding to Formula I, startingwith 11α-hydroxylation of androstendione or other compound of FormulaXXXV

wherein —A—A—, R³, and —B—B— are as defined in Formula XIII, producingan intermediate corresponding to the Formula XXXVI

where —A—A—, R³, and —B—B— are as defined in Formula XIII. Except forthe selection of substrate, the process for conducting the11α-hydroxylation is essentially as described hereinabove for Scheme 1.The following microorganisms are capable of carrying out the11α-hydroxylation of androstendione or other compound of Formula XXXV:

Aspergillus ochraceus NRRL 405 (ATCC 18500);

Aspergillus niger ATCC 11394;

Aspergillus nidulans ATCC 11267;

Rhizopus oryzae ATCC 11145;

Rhizopus stolonifer ATCC 6227b;

Trichothecium roseum ATCC 12519 and ATCC 8685.

11α-Hydroxyandrost-4-ene-3,17-dione, or other compound of Formula XXXVI,is next converted to 11α-hydroxy-3,4-enol ether of Formula (101):

where —A—A—, R³, and —B—B—, are as defined in Formula XIII and R¹¹ ismethyl or other lower alkyl (C₁ to C₄), by reaction with an etherifyingreagent such as trialkyl orthoformate in the presence of an acidcatalyst. To carry out this conversion, the 11α-hydroxy substrate isacidified by mixing with an acid such as, e.g., benzene sulfonic acidhydrate or toluene sulfonic acid hydrate and dissolved in a loweralcohol solvent, preferably ethanol. A trialkyl orthoformate, preferablytriethyl orthoformate is introduced gradually over a period of 5 to 40minutes while maintaining the mixture in the cold, preferably at about0° C. to about 15° C. The mixture is then warmed and the reactioncarried out at a temperature of between 20° C. and about 60° C.Preferably the reaction is carried out at 30° to 50° C. for 1 to 3hours, then heated to reflux for an additional period, typically 2 to 6hours, to complete the reaction. Reaction mixture is cooled, preferablyto 0° to 15°, preferably about 5° C., and the solvent removed undervacuum.

Using the same reaction scheme as described in Scheme 3, above, for theconversion of the compound of Formula XX to the compound of FormulaXVII, a 17-spirolactone moiety of Formula XXXIII is introduced into thecompound of Formula 101. For example, the Formula 101 substrate may bereacted with a sulfonium ylide in the presence of a base such as analkali metal hydroxide in a suitable solvent such as DMSO, to produce anintermediate corresponding to Formula 102:

where —A—A—, R³, R¹¹, and —B—B— are as defined in Formula 101. Theintermediate of Formula 102 is then reacted with a malonic acid diesterin the presence of an alkali metal alkoxide to form the five memberedspirolactone ring and produce the intermediate of Formula 103

where —A—A—, R³, R¹¹, R¹², and —B—B— are as defined in Formula XIII.Finally, the compound of Formula 103 in a suitable solvent, such asdimethylformamide, is subjected to heat in the presence of an alkalimetal halide, splitting off the alkoxycarbonyl moiety and producing theintermediate of Formula 104:

where again —A—A—, R³, R¹¹ and —B—B— are as defined in Formula XIII.

Next the 3,4-enol ether compound 104 is converted to the compound ofFormula XXIII, i.e., the compound of Formula VIII in which R⁸ and R⁹together form the moiety of Formula XXXIII. This oxidation step iscarried out in essentially the same manner as the oxidation step forconversion of the compound of Formula XXIV to the intermediate ofFormula XXIII in the synthesis of Scheme 4. Direct oxidation can beeffected using a reagent such as2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) ortetrachlorobenzoquinone (chloranil), or preferably a two stage oxidationis effected by first brominating, e.g., with an N-halo brominating agentsuch as N-bromosuccinamide or 1,3-dibromo-5,5-dimethyl hydantoin (DBDMH)and then dehydrobrominating with a base, for example with DABCO in thepresence of LiBr and heat. Where NBS is used for bromination, an acidmust also be employed to convert 3-enol ether to the enone. DBDMH, anionic rather than free radical bromination reagent, is effective byitself for bromination and conversion of the enol ether to the enone.

The compound of Formula VIII is then converted to epoxymexrenone orother compound of Formula I by the steps described hereinabove forScheme 1.

Each of the intermediates of Formulae 101, 102, 103, and 104 is a novelcompound having substantial value as an intermediate for epoxymexrenoneor other compounds of Formulae IA and I. In each of the compounds ofFormulae 101, 102, 103, and 104 —A—A— and —B—B— are preferably —CH₂—CH₂—and R³ is hydrogen, lower alkyl or lower alkoxy. Most preferably, thecompound of Formula 101 is 3-ethoxy-11α-hydroxyandrost-3,5-dien-17-one,the compound of Formula 102 is3-ethoxyspiro[androst-3,5-diene-17β,2′-oxiran]-11α-ol, the compound ofFormula 103 is ethyl hydrogen3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21,21-dicarboxylate,gamma-lactone, and the compound of Formula 104 is3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21-carboxylic acid,gamma-lactone.

In a particularly preferred embodiment, the overall process of Scheme 6proceeds as follows:

SCHEME 7

Scheme 7 provides for the synthesis of epoxymexrenone and othercompounds of Formula I using a starting substrate comprisingβ-sitosterol, cholesterol, stigmasterol or other compound of FormulaXXXVII

where —A—A—, R³, and —B—B— are as defined in Formula XIII, D—D is.—CH₂—CH₂— or —CH═CH—, and each of R¹³, R¹⁴, R¹⁵ and R¹⁶ is independentlyselected from among hydrogen or C₁, to C₄ alkyl.

In the first step of the synthesis 11α-hydroxyandrostendione or othercompound of Formula XXXV is prepared by bioconversion of the compound ofFormula XXXVII. The bioconversion process is carried out substantiallyin accordance with the method described hereinabove for the11α-hydroxylation of canrenone (or other substrate of Formula XIII).

In the synthesis 11α-hydroxyandrostendione, 4-androstene-3,17-dione isinitially prepared by bioconversion of the compound of Formula XXXVII.This initial bioconversion may be carried out in the manner described inU.S. Pat. No. 3,759,791, which is expressly incorporated herein byreference. Thereafter, 4-androstene-3,17-dione is converted to11α-hydroxyandrostenedione substantially in accordance with the methoddescribed hereinabove for the 11α-hydroxylation of canrenone (or othersubstrate of Formula XIII).

The remainder of the synthesis of Scheme 7 is identical to Scheme 6. Ina particularly preferred embodiment, the overall process of Scheme 7proceeds as follows:

The methods, processes and compositions of the invention, and theconditions and reagents used therein, are further described in thefollowing examples.

EXAMPLE 1

Slants were prepared with a growth medium as set forth in Table 1

TABLE 1 Y P D A (medium for slants and plates) yeast extract 20 gpeptone 20 g glucose 20 g agar 20 g distilled water, q.s. to 1000 ml pHas is 6.7 adjust at pH 5 with H₃PO₄ 10% W/V Distribute for slants: 7.5ml in 180 × 18 mm tubes for plates (10 cm of φ) 25 ml in 200 × 20 mmtubes sterilize at 120° C. for 20 minutes pH after sterilization:5

To produce first generation cultures, a colony of Aspergillus ochraceuswas suspended in distilled water (2 ml) in a test tube; and 0.15 mlaliquots of this suspension applied to each of the slants that had beenprepared as described above. The slants were incubated for seven days at25° C., after which the appearance of the surface culture was that of awhite cottony mycelium. The reverse was pigmented in orange in the lowerpart, in yellow-orange in the upper part.

The first generation slant cultures were suspended in a sterile solution(4 ml) containing Tween 80 nonionic surfactant (3% by weight), and 0.15ml aliquots of this suspension were used to inoculate second generationslants that had been prepared with the growth medium set forth in Table2

TABLE 2 (for second generation and routine slants) malt extract 20 gpeptone 1 g glucose 20 g agar 20 g distilled water q.s. to 1000 ml pH asis 5.3 distribute in tubes (180 × 18 mm) ml 7.5 sterilize at 120° C. for20 minutes

The second generation slants were incubated for 10 days at 25° C.,producing a heavy mass of golden-colored spores; reverse pigmented inbrown orange.

A protective medium was prepared having the composition set forth inTable 3.

TABLE 3 PROTECTIVE MEDIUM Skim milk 10 g distilled water 100 ml In a 250ml flask containing 100 ml of distilled water at 50°0 C., add skim milk.Sterilize at 120° C. for 15 minutes. Cool at 33° C. and use before theday is over

Cultures from five of the second generation slants were suspended in theprotective solution (15 ml) in a 100 ml flask. The suspension wasdistributed in aliquots (0.5 ml each) among 100×10 mm tubes forlyophilization. These were pre-frozen at −70° to −80° C. in anacetone/dry ice bath for 20 minutes, then transferred immediately to adrying room pre-cooled to −40° to −50° C. The pre-frozen aliquots werelyophilized at a residual pressure of 50μ Hg and ≦−30° C. At the end ofthe lyophilization, two to three granules of sterile silica gel wereadded to each tube with moisture indicator and flame seal.

To obtain mother culture slants suitable for industrial scalefermentation, a single aliquot of lyophilized culture, which had beenprepared in the manner described above, was suspended in distilled water(1 ml) and 0.15 ml aliquots of the suspension were used to inoculateslants that had been provided with a growth medium having thecomposition set forth in Table 2. The mother slants were incubated forseven days at 25° C. At the end of incubation, the culture developed onthe slants was preserved at 40° C.

To prepare a routine slant culture, the culture from a mother slant wassuspended in a sterile solution (4 ml) containing Tween 80 (3% byweight) and the resulting suspension distributed in 0.15 ml aliquotsamong slants which had been coated with the growth medium described inTable 2. The routine slant cultures may be used to inoculate the primaryseed flasks for laboratory or industrial fermentations.

To prepare a primary seed flask culture, the culture from a routineslant, which had been prepared as described above, was removed andsuspended in a solution (10 ml) containing Tween 80 (3% by weight). A0.1 aliquot of the resulting suspension was introduced into a 500 mlbaffled flask containing a growth medium having the composition setforth in Table 4.

TABLE 4 (for primary and transformation flask culture and round bottomedflask) glucose 20 g peptone 20 g yeast autolysate 20 g distilled waterq.s to pH as is 5.2 adjust at pH 5.8 with NaOH 20% distribute in 500 mlbaffled flask 100 ml distribute in 2000 ml round bottomed flasks (3baffles) 500 ml sterilize 120° C. × 20 min. pH after sterilization about5.7

The seed flask was incubated on a rotating shaker (200 rpm, 5 cmdisplacement) for 24 hours at 28° C., thereby producing a culture in theform of pellet-like mycelia having diameters of 3 to 4 mm. Onmicroscopic observation, the seed culture was found to be a pureculture, with synnematic growth, with big hyphae and well twisted. ThepH of the suspension was 5.4 to 5.6. PMV was 5 to 8% as determined bycentrifugation (3000 rpm×5 min.).

A transformation flask culture was prepared by inoculating a growthmedium (100 ml) having the composition set forth Table 4 in a second 500ml shaker flask with biomass (1 ml) from the seed culture flask. Theresulting mixture was incubated on a rotating shaker (200 rpm, 5 cmdisplacement) for 18 hours at 28° C. The culture was examined and foundto comprise pellet like mycelia with a 3-4 mm diameter. On microscopicexamination, the culture was determined to be a pure culture, withsynnematic and filamentous growth in which the apical cells were full ofcytoplasm and the olden cells were little vacuolated. The pH of theculture suspension was 5 to 5.2 and the PMV was determined bycentrifugation to be between 10% and 15%. Accordingly, the culture wasdeemed suitable for transformation of canrenone to 11α-hydroxycanrenone.

Canrenone (1 g) was micronized to about 5μ and suspended in sterilewater (20 ml). To this suspension were added: a 40% (w/v) sterileglucose solution; a 16% (w/v) sterile solution of autolyzed yeast; and asterile antibiotic solution; all in the proportions indicated for 0hours reaction time in Table 5. The antibiotic solution had beenprepared by dissolving kanamicyn sulfate (40 mg), tetracycline HCl (40mg) and cefalexin (200 mg) in water (100 ml). The steroid suspension,glucose solution, and autolyzed yeast solution were added gradually tothe culture contained in the shaker flask.

TABLE 5 Indicative Additions of Steroid and Solutions (additives andantibiotics) in the Course of Bioconversion of Canrenone in Shake Flaskyeast auto- anti- Reaction Steroid Suspension glucose lised biotic timeapprox. solution sol. solution hours ml mg. ml ml. ml  0 1  50 1 0.5 1 8 2 100 2 1 24 2 100 1 0.5 1 32 5 250 2 1 48 2 100 i 0.5 1 56 5 250 2 172 3 150 1 0.5 1 90

As reaction proceeded, the reaction mixture was periodically analyzed todetermine glucose content, and by thin layer chromatography to determineconversion to 11α-hydroxycanrenone. Additional canrenone substrate andnutrients were added to the fermentation reaction mixture during thereaction at rates controlled to maintain the glucose content in therange of about 0.1% by weight. The addition schedule for steroidsuspension, glucose solution, autolyzed yeast solution and antibioticsolution is set forth in Table 5. The transformation reaction continuedfor 96 hours at 25° C. on a rotary shaker (200 rpm and 5 cmdisplacement). The pH ranged between 4.5 and 6 during the fermentation.Whenever the PMV rose to or above 60%, a 10 ml portion of broth culturewas withdrawn and replaced with 10 ml distilled water. The disappearanceof canrenone and appearance of 11α-hydroxycanrenone were monitoredduring the reaction by sampling the broth at intervals of 4, 7, 23, 31,47, 55, 71, 80, and 96 hours after the start of the fermentation cycle,and analyzing the sample by TLC. The progress of the reaction asdetermined from these samples is set forth in Table 6

TABLE 6 Time Course of Bioconversion of Canrenone in Shake FlaskTransformation Ratio Time Canrenone Rf. 11αhydroxy Canrenone hours RF. =0.81 RF. = 0.29  0 100 0.0  4 50 50  7 20 80 23 20 80 31 30 70 47 20 8055 30 70 71 25 75 80 15 85 96 ˜10 ˜90

EXAMPLE 2

A primary seed flask culture was prepared in the manner described inExample 1. A nutrient mixture was prepared having the composition setforth in Table 7

TABLE 7 For Transformation Culture in 10 l glass fermenter quantity g/lglucose 80 g 20 peptone 80 g 20 yeast autolised 80 g 20 antifoam SAG 4710.5 g deionized water q.s. to 4 l sterilize the empty fermenter for 30minutes at 130° C. load it with 3 l of deionized water, heat at 40° C.add while stirring the components of the medium stir for 15 minutes,bring to volume of 3.9 l pH as is 5.1 adjust of 5.8 with NaOH 20% w/vsterilize at 120° C. × 20 minutes pH after sterilization 5.5 5.7

An initial charge of this nutrient mixture (4 L) was introduced into atransformation fermenter of 10 L geometric volume. The fermenter was ofcylindrical configuration with a height to diameter ratio of 2.58. Itwas provided with a 400 rpm turbine agitator having two No. 2 diskwheels with 6 blades each. The external diameter of the impellers was 80mm, each of the blades was 25 mm in radial dimension and 30 mm high, theupper wheel was positioned 280 mm below the top of the vessel, the lowerwheel was 365 mm below the top, and baffles for the vessel were 210 mmhigh and extended radially inwardly 25 mm from the interior verticalwall of the vessel.

Seed culture (40 ml) was mixed with the nutrient charge in thefermenter, and a transformation culture established by incubation for 22hours at 28° C., and an aeration rate of 0.5 l/l-min. at a pressure of0.5 kg/cm². At 22 hours, the PMV of the culture was 20-25% and the pH 5to 5.2.

A suspension was prepared comprising canrenone (80 g) in sterile water(400 ml), and a 10 ml portion added to the mixture in the transformationfermenter. At the same time a 40% (w/v) sterile glucose solution, a 16%(w/v) sterile solution of autolyzed yeast, and a sterile antibioticsolution were added in the proportions indicated in Table 8 at 0 hoursreaction time. The antibiotic solution was prepared in the mannerdescribed in Example 1.

TABLE 8 Indicative Additions of Steroid and Solutions (additives andantibiotics) in the Course of Bioconversion of Canrenone in 10 l GlassFermenter Steroid yeast anti- Reaction Suspension glucose autolisedbiotic time approx solution solution solution hours ml gr ml ml ml  0 104 25 12.5 40  4 25 12.5  8 10 4 25 12.5 12 25 12.5 16 10 4 25 12.5 20 2512.5 24 10 4 25 12.5 40 28 10 4 25 12.5 32 12.5 5 25 12.5 36 12.5 5 2512.5 40 12.5 5 25 12.5 44 12.5 5 25 12.5 48 12.5 5 25 12.5 40 52 12.5 525 12.5 56 12.5 5 25 12.5 60 12.5 5 25 12.5 64 12.5 5 25 12.5 68 12.5 525 12.5 72 12.5 5 25 12.5 40 76 12.5 5 25 12.5 80 84 88

As reaction proceeded, the reaction mixture was periodically analyzed todetermine glucose content, and by thin layer chromatography to determineconversion to 11α-hydroxycanrenone. Based on TLC analysis of reactionbroth samples as described hereinbelow, additional canrenone was addedto the reaction mixture as canrenone substrate was consumed. Glucoselevels were also monitored and, whenever glucose concentration droppedto about 0.05% by weight or below, supplemental glucose solution wasadded to bring the concentration up to about 0.25% by weight. Nutrientsand antibiotics were also added at discrete times during the reactioncycle. The addition schedule for steroid suspension, glucose solution,autolyzed yeast solution and antibiotic solution is set forth in Table8. The transformation reaction continued for 90 hours at an aerationrate of 0.5 vol. air per vol. liquid per minute (vvm) at a positive headpressure of 0.3 kg/cm². The temperature was maintained at 28° C. untilPVM reached 45%, then decreased to 26° C. and maintained at thattemperature as PVM grew from 45% to 60%, and thereafter controlled at24° C. The initial agitation rate was 400 rpm, increasing to 700 rpmafter 40 hours. The pH was maintained at between 4.7 and 5.3 byadditions of 2M orthophosphoric acid or 2M NaOH, as indicated. Foamingwas controlled by adding a few drops of Antifoam SAG 471 as foamdeveloped. The disappearance of canrenone and appearance of11α-hydroxycanrenone were monitored at 4 hour intervals during thereaction by TLC analysis of broth samples. When most of the canrenonehad disappeared from the broth, additional increments were added.

After all canrenone additions had been made, the reaction was terminatedwhen TLC analysis showed that the concentration of canrenone substraterelative to 11α-hydroxycanrenone product had dropped to about 5%.

At the conclusion of the reaction cycle, the fermentation broth wasfiltered through cheese cloth for separation of the mycelium from theliquid broth. The mycelia fraction was resuspended in ethyl acetateusing about 65 volumes (5.2 liters) per gram canrenone charged over thecourse of the reaction. The suspension of mycelia in ethyl acetate wasrefluxed for one hour under agitation, cooled to about 200° C., andfiltered on a Buchner. The mycelia cake was washed sequentially withethyl acetate (5 vol. per g canrenone charge; 0.4 L) and deionized water(500 ml) to displace the ethyl acetate extract from the cake. The filtercake was discarded. The rich extract, solvent washing and water washingwere collected in a separator, then allowed to stand for 2 hours forphase separation.

The aqueous phase was then discarded and the organic phase concentratedunder vacuum to a residual volume of 350 ml. The still bottoms werecooled to 15° C. and kept under agitation for about one hour. Theresulting suspension was filtered to remove the crystalline product, andthe filter cake was washed with ethyl acetate (40 ml). After drying, theyield of 11α-hydroxycanrenone was determined to be 60 g.

EXAMPLE 3

A spore suspension was prepared from a routine slant in the mannerdescribed in Example 1. In a 2000 ml baffled round bottomed flask (3baffles, each 50 mm×30 mm), an aliquot (0.5 ml) of the spore suspensionwas introduced into a nutrient solution (500 ml) having the compositionset forth in Table 4. The resulting mixture was incubated in the flaskfor 24 hours at 25° C. on an alternating shaker (120 strokes per min.;displacement 5 cm), thereby producing a culture which, on microscopicexamination, was observed to appear as a pure culture with hyphae welltwisted. The pH of the culture was between about 5.3 and 5.5, and thePMV (as determined by centrifugation at 3000 rpm for 5 min.) was 8 to10%.

Using the culture thus prepared, a seed culture was prepared in astainless steel fermenter of vertical cylindrical configuration, havinga geometric volume of 160 L and an aspect ratio of 2.31 (height=985 mm;diameter=425 mm). The fermenter was provided with a disk turbine typeagitator having two wheels, each wheel having six blades with anexternal diameter of 240 mm, each blade having a radial dimension of 80mm and a height of 50 mm. The upper wheel was positioned at a depth of780 mm from the top of the fermenter, and the second at a depth of 995mm. Vertical baffles having a height of 890 mm extended radiallyinwardly 40 mm from the interior vertical wall of the fermenter. Theagitator was operated at 170 rpm. A nutrient mixture (100 L) having thecomposition set forth in Table 9 was introduced into the fermenter,followed by a portion of preinoculum (1 L) prepared as described aboveand having a pH of 5.7.

TABLE 9 For Vegetative Culture in 160 L Fermenter About 8 L are neededto Seed Productive fermenter Quantity g/L glucose 2 kg 20 peptone 2 kg20 yeast autolysed 2 kg 20 antifoam SAG 471 0.010 Kg traces deionizedwater q.s. to 100 L sterilize the empty fermenter for 1 hour at 130° C.load it with 6 L of deionized water; heat at 40° C. add while stirringthe components of the medium stir for 15 minutes, bring to volume of 95L sterilization at 121° C. for 30 minutes post sterilization pH is 5.7add sterile deionized water to 100 L

The inoculated mixture was incubated for 22 hours at an aeration rate of0.5 L/L-min. at a head pressure of 0.5 kg/cm². The temperature wascontrolled at 280° C. until PMV reached 25%, and then lowered to 250° C.The pH was controlled in the range of 5.1 to 5.3. Growth of myceliumvolume is shown in Table 10, along with pH and dissolved oxygen profilesof the seed culture reaction.

TABLE 10 Time Course for Mycelial Growth in Seed Culture Fermentationpacked mycelium volume (pmv) % Fermentation (3000 rpms dissolved periodh pH 5 min) oxygen %  0 5.7 ± 0.1 100  4 5.7 ± 0.1 100  8 5.7 ± 0.1 12 ±3 85 ± 5 12 5.7 ± 0.1 15 ± 3 72 ± 5 16 5.5 ± 0.1 25 ± 5 40 ± 5 20 5.4 ±0.1 30 ± 5 35 ± 5 22 5.3 ± 0.1 33 ± 5 30 ± 5 24 5.2 ± 0.1 35 ± 5 25 ± 5

Using the seed culture thus produced, a transformation fermentation runwas carried out in a vertical cylindrical stainless steel fermenterhaving a diameter of 1.02 m, a height of 1.5 m and a geometric volume of1.4 m³. The fermenter was provided with a turbine agitator having twoimpellers, one positioned 867 cm below the top of the reactor and theother positioned 1435 cm from the top. Each wheel was provided with sixblades, each 95 cm in radial dimension and 75 cm high. Vertical baffles1440 cm high extended radially inwardly 100 cm from the interiorvertical wall of the reactor. A nutrient mixture was prepared having thecomposition set forth in Table 11

TABLE 11 For Bioconversion Culture in 1000 L Fermenter Quantity g/Lglucose 16 kg 23 peptone 16 kg 23 yeast autolysed 16 kg 23 antifoam SAG471 0.080 Kg traces deionized water q.s. to 700 L sterilize the emptyfermenter for 1 hour at 130° C. load it with 600 L ofdeionized water;heat at 40° C. add while stirring the components of the medium stir for15 minutes, bring to volume of 650 L sterilization at 121° C. for 30minutes post sterilization pH is 5.7 add sterile deionized water to 700L

An initial charge (700 L) of this nutrient mixture (pH=5.7) wasintroduced into the fermenter, followed by the seed inoculum of thisexample (7 L) prepared as described above.

The nutrient mixture containing inoculum was incubated for 24 hours atan aeration rate of 0.5 L/L-min at a head pressure of 0.5 kg/cm². Thetemperature was controlled at 28° C., and the agitation.rate was 110rpm. Growth of mycelium volume is shown in Table 12, along with pH anddissolved oxygen profiles of the seed culture reaction.

TABLE 12 Time Course for Mycelial Growth in Fermenter of theTransformation Culture packed mycelium volume (pmv) % Fermentation (3000rpm × dissolved period h pH 5 min) oxygen %  0 5.6 ± 0.2 100  4 5.5 ±0.2 100  8 5.5 ± 0.2 12 ± 3 95 ± 5 12 15 ± 3 90 ± 5 16 5.4 ± 0.1 20 ± 575 ± 5 20 5.3 ± 0.1 25 ± 5 60 ± 5 22 5.2 ± 0.1 30 ± 5 40 ± 5

At the conclusion of the incubation, pelleting of the mycelium wasobserved, but the pellets were generally small and relatively looselypacked. Diffuse mycelium was suspended in the broth. Final pH was 5.1 to5.3.

To the transformation culture thus produced was added a suspension ofcanrenone (1.250 kg; micronized to 5μ) in sterile water (5 L). Sterileadditive solution and antibiotic solution were added in the proportionsindicated at reaction time 0 in Table 14. The composition of theadditive solution is set forth in Table 13.

TABLE 13 ADDITIVE SOLUTION (for transformative culture) quantitydextrose 40 Kg yeast autolysate 8 Kg antifoam SAG 471 0.010 Kg deionizedwater q.s. to 100 l sterilize a 150 l empty fermenter for 1 hour at 130°C. load it with 70 l of deionized water; heat at 40° C. add whilestirring the components of “additive solution” stir for 30 minutes,bring to volume of 95 l pH as is 4.9 sterilize at 120° C. × 20 minutespH after sterilization about 5

Bioconversion was.carried out for about 96 hours with aeration at 0.5L/L-min. at a head pressure of 0.5 kg/cm² and a pH of ranging between4.7 and 5.3, adjusted as necessary by additions of 7.5 M NaOH or 4 MH₃PO₄. The agitation rate was initially 100 rpm, increased to 165 rpm at40 hours and 250 rpm at 64 hours. The initial temperature was 28° C.,lowered to 26° C. when PMV reached 45%, and lowered to 24° C. when PMVrose to 60%. SAG 471 in fine drops was added as necessary to controlfoaming. Glucose levels in the fermentation were monitored at 4 hourintervals and, whenever the glucose concentration fell below 1 gpl, anincrement of sterile additive solution (10 L) was added to the batch.Disappearance of canrenone and appearance of 11α-hydroxycanrenone werealso monitored during the reaction by HPLC. When at least 90% of theinitial canrenone charge had been converted to 11α-hydroxycanrenone, anincrement of 1.250 kg canrenone was added. When 90% of the canrenone inthat increment was shown to have been converted, another 1.250 kgincrement was introduced. Using the same criterion further increments(1.250 kg apiece) were added until the total reactor charge (20 kg) hadbeen introduced. After the entire canrenone charge had been delivered tothe reactor, reaction was terminated when the concentration of unreactedcanrenone was 5% relative to the amount of 11α-hydroxycanrenoneproduced. The schedule for addition of canrenone, sterile additivesolution, and antibiotic solution is as shown in Table 14.

TABLE 14 Additions of the Steroid and Solutions (additives andantibiotics) in the Course of Bioconversion of Canrenone in FermenterCANRENONE Sterile anti- Reaction in suspension additive biotic volumetime Progress solution solution liters hours Kg -ive Kg liters litersabout  0 1.250 1.25 10 8 700  4 10  8 1.250 2.5 10 12 10 16 1.250 10 2010 24 1.250 10 8 800 28 1.250 10 32 1.250 10 36 1.250 10 40 1.250 10 441.250 10 48 1.250 12.5 10 8 900 52 1.250 10 56 1.260 10 60 1.250 10 641.250 10 68 1.250 10 72 1.250 20 10 8 1050 76  0 80 84 88 92 Total

When bioconversion was complete, the mycelia were separated from thebroth by centrifugation in a basket centrifuge. The filtrate wasdetermined by HPLC to contain only 2% of the total quantity of11α-hydroxycanrenone in the harvest broth, and was therefore eliminated.The mycelia were suspended in ethyl acetate (1000 L) in an extractiontank of 2 m³ capacity. This suspension was heated for one hour underagitation and ethyl acetate reflux conditions, then cooled andcentrifuged in a basket centrifuge. The mycelia cake was washed withethyl acetate (200 L) and thereafter discharged. The steroid richsolvent extract was allowed to stand for one hour for separation of thewater phase. The water phase was extracted with a further amount ofethyl acetate solvent (200 L) and then discarded. The combined solventphases were clarified by centrifugation and placed in a concentrator(500 L geometric volume) and concentrated under vacuum to a residualvolume of 100 L. In carrying out the evaporation, the initial charge tothe concentrator of combined extract and wash solutions was 100 L, andthis volume was kept constant by continual or periodic additions ofcombined solution as solvent was taken off. After the evaporation stepwas complete, the still bottoms were cooled to 20° C. and stirred fortwo hours, then filtered on a Buchner filter. The concentrator pot waswashed with ethyl acetate (20 L) and this wash solution was then used towash the cake on the filter. The product was dried under vacuum for 16hours at 50° C. Yield of 11α-hydroxycanrenone was 14 kg.

EXAMPLE 4

Lyophilized spores of Aspergillus ochraceus NRRL 405 were suspended in acorn steep liquor growth medium (2 ml) having the composition set forthin Table 15:

TABLE 15 Corn Steep Liquor Medium (Growth Medium for Primary SeedCultivation) Corn steep liquor 30 g Yeast extract 15 g Ammoniumphosphate  3 g Monobasic Glucose (charge after sterilization) 30 gdistilled water, q.s. to 1000 ml pH as is: 4.6, adjust to pH 6.5 with20% NaOH, distribute 50 ml to 250 ml Erlenmeyer flask sterilize 121° C.for 20 minutes.

The resulting suspension was used in an inoculum for the propagation ofspores on agar plates. Ten agar plates were prepared, each bearing asolid glucose/yeast extract/phosphate/agar growth medium having thecomposition set forth in Table 16:

TABLE 16 GYPA (Glucose/Yeast Extract/Phosphate Agar for Plates) Glucose(charge after sterilization) 10 g Yeast extract 2.5 g K₂HPO₄ 3 g Agar 20g distilled water, q.s. to 1000 ml adjust pH to 6.5 sterilize 121° C.for 30 minutes

A 0.2 ml aliquot of the suspension was transferred onto the surface ofeach plate. The plates were incubated at 25° C. for ten days, afterwhich the spores from all the plates were harvested into a sterilecryogenic protective medium having the composition set forth in Table17:

TABLE 17 GYP/Glycerol (Glucose/Yeast Extract/ Phosphate/Glycerol mediumfor stock vials) Glucose (charge after sterilization) 10 g Yeast extract2.5 g K₂HPO₄ 3 g Glycerol 20 g Distilled water, q.s. to 1000 mLSterilize at 121° C. for 30 minutes

The resulting suspension was divided among twenty vials, with one mlbeing transferred to each vial. These vials constitute a master cellbank that can be drawn on to produce working cell banks for use ingeneration of inoculum for bioconversion of canrenone to11α-hydroxycanrenone. The vials comprising the master cell bank werestored in the vapor phase of a liquid nitrogen freezer at −130° C.

To begin preparation of a working cell bank, the spores from a singlemaster cell bank vial were resuspended in a sterile growth medium (1 ml)having the composition set forth in Table 15. This suspension wasdivided into ten 0.2 ml aliquots and each aliquot used to inoculate anagar plate bearing a solid growth medium having the composition setforth in Table 16. These plates were incubated for ten days at 25° C. Bythe third day of incubation, the underside of the growth medium wasbrown-orange. At the end of the incubation there was heavy production ofgolden colored spores. The spores from each plate were.harvested by theprocedure described hereinabove for the preparation of the master cellbank. A total of one hundred vials was prepared, each containing 1 ml ofsuspension. These vials constituted the working cell bank. The workingcell bank vials were also preserved by storage in the vapor phase of aliquid nitrogen freezer at −130° C.

Growth medium (50 ml) having the composition set forth in Table 15 wascharged to a 250 ml Erlenmeyer flask. An aliquot (0.5 ml) of workingcell suspension was introduced into the flask and mixed with the growthmedium. The inoculated mixture was incubated for 24 hours at 25° C. toproduce a primary seed culture having a percent packed mycelial volumeof approximately 45%. Upon visual inspection the culture was found tocomprise pellet-like mycelia of 1 to 2 mm diameter; and upon microscopicobservation it appeared as a pure culture.

Cultivation of a secondary seed culture was initiated by introducing agrowth medium having the composition set forth in Table 15 into a 2.8 LFernbach flask, and inoculating the medium with a portion (10 ml) of theprimary seed culture of this example, the preparation of which was asdescribed above. The inoculated mixture was incubated at 25° C. for 24hours on a rotating shaker (200 rpm, 5 cm displacement). At the end ofthe incubation, the culture exhibited the same properties as describedabove for the primary seed culture, and was suitable for use in atransformation fermentation in which canrenone was bioconverted to11α-hydroxycanrenone.

Transformation was conducted in a Braun E Biostat fermenter configuredas follows:

Capacity: 15 liters with round bottom Height: 53 cm Diameter: 20 cm H/D:2.65 Impellers: 7.46 cm diameter, six paddles 2.2 × 1.4 cm each Impellerspacing: 65.5, 14.5 and 25.5 cm from bottom of tank Baffles: four 1.9 ×48 cm Sparger: 10.1 cm diameter, 21 holes −1 mm diameter Temperaturecontrol: provided by means of an external vessel jacket

Canrenone at a concentration of 20 g/L was suspended in deionized water(4 L) and a portion (2 L) of growth medium having the composition setforth in Table 18 was added while the mixture in the fermenter wasstirred at 300 rpm.

TABLE 18 (Growth medium for bioconversion culture in 10 L fermenter)Quantity Amount/L glucose (charge after 160 g 20 g sterilization)peptone 160 g 20 g yeast extract 160 g 20 g antifoam SAF471 4.0 ml 0.5ml Canrenone 160 g 20 g deionized water q.s. to 7.5 L sterilize 121° C.for 30 minutes

The resulting suspension was stirred for 15 minutes, after which thevolume was brought up to 7.5 L with additional deionized water. At thispoint the pH of the suspension was adjusted from 5.2 to 6.5 by additionof 20% by weight NaOH solution, and the suspension was then sterilizedby heating at 121° C. for 30 minutes in the Braun E fermenter. The pHafter sterilization was 6.3±0.2, and the final volume was 7.0 L. Thesterilized suspension was inoculated with a portion (0.5 L) of thesecondary seed culture of this example that has been prepared asdescribed above, and the volume brought up to 8.0 L by addition of 50%sterile glucose solution. Fermentation was carried out at a temperatureof 28° C. until the PMV reached 50%, then lowered to 26° C., and furtherlowered to 24° C. when PMV exceeded 50% in order to maintain aconsistent PMV below about 60%. Air was introduced through the spargerat a rate of 0.5 vvm based on initial liquid volume and the pressure inthe fermenter was maintained at 700 millibar gauge. Agitation began at600 rpm and was increased stepwise to 1000 rpm as needed to keep thedissolved oxygen content above 30% by volume. Glucose concentration wasmonitored. After the initial high glucose concentration fell below 1%due to consumption by the fermentation reaction, supplemental glucosewas provided via a 50% by weight sterile glucose solution to maintainthe concentration in the 0.05% to 1% range throughout the remainder ofthe batch cycle. Prior to inoculation the pH was 6.3±0.2. After the pHdropped to about 5.3 during the initial fermentation period, it wasmaintained in the range of 5.5±0.2 for the remainder of the cycle byaddition of ammonium hydroxide. Foam was controlled by adding apolyethylene glycol antifoam agent sold under the trade designation SAG471 by OSI Specialties, Inc.

Growth of the culture took place primarily during the first 24 hours ofthe cycle, at which time the PMV was about 40%, the pH was about 5.6 andthe dissolved oxygen content was about 50% by volume. Canrenoneconversion began even as the culture was growing. Concentrations ofcanrenone and 11α-hydroxycanrenone were monitored during thebioconversion by analyzing daily samples. Samples were extracted withhot ethyl acetate and the resulting sample solution analyzed by TLC andHPLC. The bioconversion was deemed complete when the residual canrenoneconcentration was about 10% of the initial concentration. Theapproximate conversion time was 110 to 130 hours.

When bioconversion was complete, mycelial biomass was separated from thebroth by centrifugation. The supernatant was extracted with an equalvolume of ethyl acetate, and the aqueous layer discarded. The mycelialfraction was resuspended in ethyl acetate using approximately 65 volumesper g canrenone charged to the fermentation reactor. The mycelialsuspension was refluxed for one hour under agitation, cooled to about20° C., and filtered on a Buchner funnel. The mycelial filter cake waswashed twice with 5 volumes of ethyl acetate per g of canrenone chargedto the fermenter, and then washed with deionized water (1 L) to displacethe residual ethyl acetate. The aqueous extract, rich solvent, solventwashing and water washing were combined. The remaining exhaustedmycelial cake was either discarded or extracted again, depending onanalysis for residual steroids therein. The combined liquid phases wereallowed to settle for two hours. Thereafter, the aqueous phase wasseparated and discarded, and the organic phase concentrated under vacuumuntil the residual volume was approximately 500 ml. The still bottle wasthen cooled to about 15° C. with slow agitation for about one hour. Thecrystalline product was recovered by filtration, and washed with chilledethyl acetate (100 ml). Solvent was removed from the crystals byevaporation, and the crystalline product dried under vacuum at 50° C.

EXAMPLE 5

Lyophilized spores of Asiergillus ochraceus ATCC 18500 were suspended ina corn steep liquor growth medium (2 ml) as described in Example 4. Tenagar plates were prepared, also in the manner of Example 4. The plateswere incubated and harvested as described in Example 4 to provide amaster cell bank. The vials comprising the master cell bank were storedin the vapor phase of a liquid nitrogen freezer at −130° C.

From a vial of the master cell bank, a working cell bank was prepared asdescribed in Example 4, and stored in the nitrogen freezer at −130° C.

Growth medium (300 mL) having the composition set forth in Table 19 wascharged to a 2 L baffled flask. An aliquot (3 mL) of working cellsuspension was introduced into the flask. The inoculated mixture wasincubated for 20 to 24 hours at 28° C. on a rotating shaker (200 rpm, 5cm displacement) to produce a primary seed culture having a percentpacked mycelial volume of approximately 45%. Upon visual inspection theculture was found to comprise pellet like mycelia of 1 to 2 mm diameter;and upon microscopic observation it appeared as a pure culture.

TABLE 19 Growth medium for primary and secondary seed cultivationAmount/L glucose (charge after 20 g sterilization) peptone 20 g Yeastextract 20 g distilled water q.s. to 1000 mL sterilize 121° C. for 30minutes

Cultivation of a secondary seed culture was initiated by introducing 8 Lgrowth medium having the composition set forth in Table 19 into a 14 Lglass fermenter. Inoculate the fermenter with 160 mL to 200 mL of theprimary seed culture of this example. The preparation of which was asdescribed above.

The inoculated mixture was cultivated at 28° C. for 18-20 hours, 200 rmpagitation, aeration rate was 0.5 vvm. At the end of the propagation, theculture exhibited the same properties as described above for the primaryseed.

Transformation was conducted in a 60 L fermenter, substantially in themanner described in Example 4, except that the growth medium had thecomposition set forth in Table 20, and the initial charge of secondaryseed culture was 350 mL to 700 mL. Agitation rate was initially 200 rpm,but increased to 500 rpm as necessary to maintain dissolved oxygen above10% by volume. The approximate bioconversion time for 20 g/L canrenonewas 80 to 160 hours.

TABLE 20 Growth Medium for Bioconversion Culture in 60 L FermenterQuantity Amount/L glucose (charge after 17.5 g 0.5 g sterilization)peptone 17.5 g 0.5 g yeast extract 17.5 g 0.5 g Canrenone (charge as a700 g 20 g 20% slurry in sterile water) deionized water, q.s. to 35 Lsterilize 121° C. for 30 minutes

EXAMPLE 6

Using a spore suspension from the working cell bank produced inaccordance with the method described in Example 4, primary and secondaryseed cultures were prepared, also substantially in the manner describedin Example 4. Using secondary seed culture produced in this manner, twobioconversion runs were made in accordance with a modified process ofthe type illustrated in FIG. 1, and two runs were made with the processillustrated in FIG. 2. The transformation growth medium, canrenoneaddition schedules, harvest times, and degrees of conversion for theseruns are set forth in Table 21. Run R2A used a canrenone addition schemebased on the same principle as Example 3, while run R2C modified theExample 3 scheme by making only two additions of canrenone, one at thebeginning of the batch, and one after 24 hours. In runs R2B and R2D, theentire canrenone charge was introduced at the beginning of the batch andthe process generally carried in the manner described in Example 4,except that the canrenone charge was sterilized in a separate vesselbefore it was charged to the fermenter and glucose was added as thebatch progressed. A Waring blender was used to reduce chunks produced onsterilization. In runs R2A and R2B, canrenone was introduced into thebatch in methanol solution, in which respect these runs further differedfrom the runs of Examples 3 and 4, respectively.

TABLE 21 Descriptions of the Initial Canrenone Bioconversion ProcessesRun Number R2A R2B R2C R2D Medium (g/L) Corn steep liq. 30 the same asrun 30 the same as run Yeast extract 15 R2A 15 R2C NH₄H₂PO₄ 3 3 Glucose15 30 OSA 0.5 ml 0.5 ml pH adjusted to 6.0 adjusted to with 2.5 NNaOH6.5 with 2.5 NNaOH Canrenone 10 g/80 ml MEOH 80 g/640 ml MEOH Sterilizedand Sterilized and added at 0, 18, added at 0 hr all blended; addedblended; added 24, 30, 36, 42, at once at: 0 hr: 25 g at: 0 hr: 200 g50, 56, 62 and 24 hr: 200 g 68 hr. Harvest time 143 hrs. 166 hrs. 125hrs. 104 hrs. Bioconversion 45.9% 95.6% 98.1% 95.1%

In runs R2A and R2B, the methanol concentration accumulated to about6.0% in the fermentation beer, which was found to be inhibitory to thegrowth of culture and bioconversion. However, based on the results ofthese runs, it was concluded that methanol.or other water-misciblesolvent could serve effectively at lower concentrations to increase thecanrenone charge and provide canrenone as a fine particle precipitateproviding a large interfacial area for supply of canrenone to thesubject to the reaction.

Canrenone proved stable at sterilization temperature (121° C.) butaggregated into chunks. A Waring blender was employed to crush the lumpsinto fine particles, which were successfully converted to product.

EXAMPLE 7

Using a spore suspension from the working cell bank produced inaccordance with the method described in Example 4, primary and secondaryseed cultures were prepared, also substantially in the manner describedin Example 4. The description and results of Example 7 are shown inTable 22. Using secondary seed culture produced in this manner, onebioconversion (R3C) was carried out substantially as described inExample 3, and three bioconversions were carried out in accordance withthe process generally described in Example 5. In the latter three runs(R3A, R3B and R3D), canrenone was sterilized in a portable tank,together with the growth medium except for glucose. Glucose wasaseptically fed from another tank. The sterilized canrenone suspensionwas introduced into the fermenter either before inoculation or duringthe early stage of bioconversion. In run R3B, supplemental sterilecanrenone and growth medium was introduced at 46.5. Lumps of canrenoneformed on sterilization were delumped through a Waring blender thusproducing a fine particulate suspension entering the fermenter. Thetransformation growth media, canrenone addition schedules, nutrientaddition schedules, harvest times, and degrees of conversion for theseruns are set forth in Tables 22 and 23.

TABLE 22 Descriptions of Process for Canrenone Bioconversion Run NumberR3A R3B R3C R3D Medium (g/L) Corn steep liq. 30 the same as run Peptone:20 the same as run Yeast extract 15 R3A Yeast Ext.: 20 R3A NH₄H₂PO₄ 3Glucose: 20 Glucose 15 OSA: 3 ml OSA 0.5 ml pH adjusted to 6.5 adjustedto 6.5 with 2.5 N NaOH with 2.5 NNaOH Canrenone canrenone was the sameas run Non-sterile The same as run charge at sterilized and R3Acanrenone: R3A blended. BI: 50 g BI: 50 g charged by the 16.5 hrs: 110 g16.5 hrs: 110 g scheduled listed BI: 50 g 46.5 hrs: 80 g in Table 2316.5 hrs: 110 g Feedings see Table 23 see Table 23 see Table 23 seeTable 23 Harvest time 118.5 hrs. 118.5 hrs. 118.5 hrs. 73.5 hrs.Bioconversion 93.7% 94.7% 60.0% 68.0%

TABLE 23 The Feeding Schedule for Canrenone, Glucose and Growth Mediumin the Development Experiment R3C Antibiotics R3A R3B R3D Peptone & 20mg kanamycin Canrenone/ Canrenone/ Canrenone/ canrenone Yeast ext. 20 mgGrowth Growth Growth 200 g/2 L 20 g each tetracycline Medium MediumMedium sterile Glucose 50% in IL 100 mg cefalexin see see see AdditionDI solution water in 50 ml Table 22 Table 22 Table 22 Time hr. g g g in50 ml g/L g/L g/L 0 — — — —  50 g/0.4 L  50 g/0.4 L  50 g/0.4 L 14.5  16100 25 50 ml — — — 16.5 — — — — 100 g/1.2 L 110 g/1.2 L 100 g/1.2 L 20.5 16 140 25 — — — — 28.5  16 140 25 — — — — 34.5  16 150 25 — — — — 40.5 16 150 25 50 ml — — — 46.5 880 130 25 — — 80 g/0.8 L — 52.5 160 120 25— — — — 58.5 160 150 25 — — — — 64.5 160 180 25 50 ml — — — 70.5 160 14025 — — — —

Due to filamentous growth, a highly viscous fermenter broth was seen inall four of the runs of this Example. To overcome obstacles which highviscosity created with respect to aeration, mixing, pH control andtemperature control, the aeration rate and agitation speed wereincreased during these runs. Conversions proceeded satisfactorily underthe more severe conditions, but a dense cake formed above the liquidbroth surface. Some unreacted canrenone was carried out of the broth bythis cake.

EXAMPLE 8

The description and results of Example 8 are summarized in Table 24.Four fermentation runs were made in which 11α-hydroxycanrenone wasproduced by bioconversion of canrenone. In two of these runs (R4A andR4D), the bioconversion was conducted in substantially the same manneras runs R3A and R3D of Example 6. In run R4C, canrenone was converted to11α-hydroxycanrenone generally in the manner described in Example 3. InRun R4B, the process was carried out generally as described in Example4, i.e., with sterilization of canrenone and growth medium in thefermenter just prior to inoculation, all nitrogen and phosphorusnutrients were introduced at the start of the batch, and a supplementalsolution containing glucose only was fed into the fermenter to maintainthe glucose level as the batch proceeded. In the latter process (runR4B), glucose concentration was monitored every 6 hours and glucosesolution added as indicated to control glucose levels in the 0.5 to 1%range. The canrenone addition schedules for these runs are set forth inTable 25.

TABLE 24 Descriptions of the Process Development Experiment of CanrenoneBioconversions Run Number R4A R4B R4C R4D Medium (g/L) Corn steep liq.30 the same as run Peptone: 20 the same as run Yeast extract 15 R4AYeast ext.: 20 R4A NH4H2PO4 3 Glucose: 20 Glucose 15 OSA 3 ml OSA 0.5 mlpH adjusted to 6.5 adjusted to 6.5 with 2.5 NNaOH with 2.5 NNaOHCanrenone Canrenone was 160 g canrenone is Nonsterile Canrenone wascharge at sterilized and sterilized in the canrenone: sterilized andblended. fermenter charged by the blended. BI: 40 g schedule listed BI:40 g 23.5 hrs: 120 g in Table 25 23.5 hrs: 120 g Medium charge see Table25 see Table 25 see Table 25 see Table 25 Harvest time 122 hrs. 122 hrs.122 hrs. 122 hrs. Bioconversion 95.6% 97.6% 95.4% 96.7%

TABLE 25 The Feeding Schedule of Canrenone, Glucose and Growth Medium inthe Development Experiment R4C Antibiotics 20 mg kanamycin Peptone & 20mg Canrenone Yeast ext. tetracycline R4A R4B R4D 200 g/2 L Glucose 20 geach 100 mg cefalexin Growth Growth Growth sterile 50% in 1 L in 50 ml(added Medium Medium Medium Addition water solution water in canrenonesee see see Time hr. g g g slurry) Table 24 Table 24 Table 24 14 600 13525 50 ml — — — 20 — 100 — — — — — 23 — — — — 120 g/1.2 L — 120 g/1.2 L26 — 100 25 — — — — 32 — 135 25 — — — — 38 500 120 25 50 ml — — — 44 —100 25 — — — — 50 — 100 25 — — — — 56 — 150 25 — — — — 62 500 150 25 50ml — — — 68 — 200 25 — — — — 74 — 300 25 — — — — 8− — 100 25 — — — — 86— 125 25 — — — — 92 — 175 25 — — — — 98 — 150 — — — — — 104 — 175 — — —— — 110 — 175 — — — — — 116 — 200 — — — — —

All fermenters were run under high agitation and aeration during most ofthe fermentation cycle because the fermentation beer had become highlyviscous within a day or so after inoculation.

EXAMPLE 9

The transformation growth media, canrenone addition schedules, harvesttimes, and degrees of conversion for the runs of this Example are setforth in Table 26.

Four bioconversion runs were carried out substantially in the mannerdescribed for run R4B of Example 8, except as described below. In runR5B, the top turbine disk impeller used for agitation in the other runswas replaced with a downward pumping marine impeller. The downwardpumping action axially poured the broth into the center of the fermenterand reduced cake formation. Methanol (200 ml) was added immediatelyafter inoculation in run R5D. Since canrenone was sterilized in thefermenter, all nutrients except glucose were added at the start of thebatch, obviating the need for chain feeding of sources of nitrogen,sources of phosphorus or antibiotics.

TABLE 26 Process Description of the Process Development Experiment of 10L Scale Bioconversions Run Number R5A R5B R5C R5D Medium (g/L) Cornsteep liq. 30 the same as run Peptone: 20 the same as run Yeast Extract15 R5A Yeast Ext.: 20 R5A NH₄H₂PO₄ 3 Glucose: 20 Glucose 15 OSA 3 ml OSA0.5 ml pH adjusted to 6.5 adjusted to 6.5 with 2.5 NNaOH with 2.5 NNaOHCanrenone 160 g canrenone 160 g canrenone 160 g canrenone 160 gcanrenone charge sterilized in the sterilized in the sterilized insterilized in fermenter fermenter the fermenter the fermenter Mediumglucose feeding glucose feeding glucose feeding glucose feeding feedingHarvest time 119.5 hrs. 119.5 hrs. 106 119.5 hrs Bioconversion 96% 94.1%88.5% 92.4%

In order to maintain immersion of the solid phase growing above theliquid surface, growth medium (2 L) was added to each fermenter 96 hoursafter the beginning of the batch. Mixing problems were not entirelyovercome by either addition of growth medium or use of a downwardpumping impeller (run R5B) but the results of the runs demonstrated thefeasibility and advantages of the process, and indicated thatsatisfactory mixing could be provided according to conventionalpractices.

EXAMPLE 10

Three bioconversion runs were carried out substantially in the mannerdescribed in Example 9. The transformation growth media, canrenoneaddition schedules, harvest times, and degrees of conversion for theruns of this Example are set forth in Table 27:

TABLE 27 Process Description of the Experiment 10 L Scale BioconversionRun Number R6A R6B R6C Medium (g/L) Corn steep liq. 30 the same as runPeptone: 20 Yeast Extract 15 R6A Yeast Ext.: 20 NH₄H₂PO₄ 3 Glucose: 20Glucose 15 OSA OSA 0.5 ml 0.5 ml pH adjusted to 6.5 adjusted to 6.5 with2.5N NaOH with 2.5 N NaOH Canrenone 160 g canrenone 160 g canrenone 160g canrenone charge sterilized in the sterilized in the sterilized infermenter fermenter the fermenter Medium glucose feeding; glucosefeeding; glucose feeding 1.3 L medium 0.5 L medium feeding; no and 0.8 Lsterile and 0.5 L sterile other addition water at 71 hrs. water at 95hrs Harvest time 120 hrs. 120 hrs. 120 hrs. Bioconversion 95% 96% 90%Mass Balance 59% 54% 80%

Growth medium (1.3 L) and sterile water (0.8 L) were added after 71hours in run R6A to submerge mycelial cake which had grown above thesurface of the liquid broth. For the same purpose, growth medium (0.5 L)and sterile water (0.5 L) were added after 95 hours in run R6B. Materialbalance data showed that a better mass balance could be determined wherecake buildup above the liquid surface was minimized.

EXAMPLE 11

Fermentation runs were made to compare pre-sterilization of canrenonewith sterilization of canrenone and growth medium in the transformationfermenter. In run R7A, the process was carried out as illustrated inFIG. 2, under conditions comparable to those of runs R2C, R2D, R3A, R3B,R3D, R4A, and R4D. Run R7B was as illustrated in FIG. 3 under conditionscomparable to those of Examples 4, 9 and 10, and run R4B. Thetransformation growth media, canrenone addition schedules, harvesttimes, and degrees of conversion for the runs of this Example are setforth in Table 28:

TABLE 28 Process Description of the Experiment of 10 L ScaleBioconversions Run Number R7A R7B Medium (g/L) corn steep liq. 30 thesame as run Yeast extract 15 R7A NH₄H₂PO₄ 3 Glucose 15 OSA 0.5 ml pHadjusted to 6.5 with 2.5 NNaOH Canrenone charge 160 g canrenone 160 gcanrenone was sterilized & was sterilized blended outside in thefermenter the fermenter Medium charge Glucose feeding; Glucose feeding;canrenone was no other added with 1.6 L addition growth medium Harvesttime 118.5 hrs. 118.5 hrs. Bioconversion 93% 89%

A mass balance based on the final sample taken from run R7B was 89.5%,indicating that no significant substrate loss or degradation inbioconversion. Mixing was determined to be adequate for both runs.

Residual glucose concentration was above the desired 5-10 gpl controlrange during the initial 80 hours. Run performance was apparentlyunaffected by a light cake that accumulated in the head space of boththe fermenters.

EXAMPLE 12

Extraction efficiency was determined in a series of 1 L extraction runsas summarized in Table 29. In each of these runs, steroids wereextracted from the mycelium using ethyl acetate (1 L/L fermentationvolume). Two sequential extractions were performed in each run. Based onRP-HPLC, About 80% of the total steroid was recovered in the firstextraction; and recovery was increased to 95% by the second extraction.A third extraction would have recovered another 3% of steroid. Theremaining 2% is lost in the supernatant aqueous phase. The extract wasdrawn to dryness using vacuum but was not washed with any additionalsolvent. Chasing with solvent would improve recovery from the initialextraction if justified by process economics.

TABLE 29 Recovery of 11α-Hydroxycanrenone at 1 Liter Extraction (% ofTotal) 1st 2nd 3rd Run Number Extract Extract Extract Supernatant R5A79% 16% 2% 2% R5A 84% 12% 2% 2% R4A 72% 20% 4% 4% R4A 79% 14% 2% 5% R4B76% 19% 4% 1% R4B 79% 16% 3% 2% R4B 82% 15% 2% 1% Average 79% 16% 3% 2%

Methyl isobutyl ketone (MIBK) and toluene were evaluated asextraction/crystallization solvents for 11α-hydroxycanrenone at the 1 Lbroth scale. Using the extraction protocol as described hereinabove,both MIBK and toluene were comparable to ethyl acetate in bothextraction efficiency and crystallization performance.

EXAMPLE 13

As part of the evaluation of the processes of FIGS. 2 and 3, particlesize studies were conducted on the canrenone substrate provided at thestart of the fermentation cycle in each of these processes. As describedabove, canrenone fed to the process of FIG. 1 was micronized beforeintroduction into the fermenter. In this process, the canrenone is notsterilized, growth of unwanted microorganisms being controlled.byaddition of antibiotics. The processes of FIGS. 2 and 3 sterilize thecanrenone before the reaction. In the process of FIG. 2, this isaccomplished in a blender before introduction of canrenone into thefermenter. In the process of FIG. 3, a suspension of canrenone in growthmedium is sterilized in the fermenter at the start of the batch. Asdiscussed hereinabove, sterilization tends to cause agglomeration ofcanrenone particles. Because of the limited solubility of canrenone inthe aqueous growth medium, the productivity of the process depends onmass transfer from the solid phase, and thus may be expected to dependon the interfacial area presented by the solid particulate substratewhich in turn depends on the particle size distribution. Theseconsiderations initially served as deterrents to the processes of FIGS.2 and 3.

However, agitation in the blender of FIG. 2 and the fermentation tank ofFIG. 3, together with the action of the shear pump used for transfer ofthe batch in FIG. 2, were found to degrade the agglomerates to aparticle size range reasonably approximate that of the unsterilized andmicronized canrenone fed to the process of FIG. 1. This is illustratedby the particle size distributions for the canrenone as available at theoutset of the reaction cycle in each of the three processes. See Table30 and FIGS. 4 and 5.

TABLE 30 Particle Distributions of Three Different Canrenone Samplesmean 45- <180 size Run #: % Sample 125 μ μ μ Bioconversion Canrenone 75%95% — R3C: shipment 93.1% (120 h) R4C: 96.3% (120 h) Blended 31.2% 77.2139.5 R3A: Sample % 94.6% (120 h) R3B: 95.2% (120 h) Sterilize 24.7%65.1 157.4 R4B: d Sample % 97.6% (120 h) R5B: 93.8% (120 h)

From the data in Table 30, it will be noted that agitators and shearpump were effective to reduce the average particle size of thesterilized canrenone to the same order of magnitude as the unsterilizedsubstrate, but a significance size difference remained in favor of theunsterilized substrate. Despite this difference, reaction performancedata showed that the pre-sterilization processes were at least asproductive as the process of FIG. 1. Further advantages may be realizedin the process of FIG. 2 by certain steps for further reducing andcontrolling particle size, e.g., wet milling of sterilized canrenone,and/or by pasteurizing rather than sterilizing.

EXAMPLE 14

A seed culture was prepared in the manner described in Example 5. At 20hours, the mycelia in the inoculum fermenter was pulpy with a 40% PMV.Its pH was 5.4 and 14.8 gpl glucose remained unused.

A transformation growth medium (35 L) was prepared having thecomposition shown in Table 20. In the preparation of feeding medium,glucose and yeast extract were sterilized separately and mixed as asingle feed at an initial concentration of 30% by weight glucose and 10%by weight yeast extract. pH of the feed was adjusted to 5.7.

Using this medium, (Table 20), two bioconversion runs were made for theconversion of canrenone to 11α-hydroxycanrenone. Each of the runs wasconducted in a 60 L fermenter provided with an agitator comprising oneRushton turbine impeller and two Lightnin' A315 impellers.

Initial charge of the growth medium to the fermenter was 35 L.Micronized and unsterilized canrenone was added to an initialconcentration of 0.5%. The medium in the fermenter was inoculated with aseed culture prepared in the manner described in Example 5 at an initialinoculation ratio of 2.5%. Fermentation was carried out at a temperatureof 28° C., an agitation rate of 200 to 500 rpm, an aeration rate of 0.5vvm, and backpressure sufficient to maintain a dissolved oxygen level ofat least 20% by volume. The transformation culture developed during theproduction run was in the form of very small oval pellets (about 1-2mm). Canrenone and supplemental nutrients were chain fed to thefermenter generally in the manner described in Example 1. Nutrientadditions were made every four hours at a ratio of 3.4 g glucose and 0.6g yeast extract per liter of broth in the fermenter.

Set forth in Table 31 are the aeration rate, agitation rate, dissolvedoxygen, PMV, and pH prevailing at stated intervals during each of theruns of this Example, as well as the glucose additions made during thebatch. Table 32 shows the canrenone conversion profile. Run R11A wasterminated after 46 hours; Run R11B continued for 96 hours. In thelatter run, 93% conversion was reached at 81 hours; one more feedaddition was made at 84 hours; and feeding then terminated. Note that asignificant change in viscosity occurred between the time feeding wasstopped and the end of the run.

TABLE 31 air Gluc cc Time (1 pm) rpm % DO Backpress PMV(%) pH (g/l)Fermentation R11A 0.1 20 200 93 0 2 6.17 5.8 7 20 200 85.1 0 5 6.03 5.512.4 20 300 50.2 0 5.43 21.8 20 400 25.5 0 38 6.98 0 29 20 500 17 0 355.22 30.2 20 500 18.8 10 5.01 31 20 500 79 10 4.81 1 35.7 20 500 100 1045 5.57 0 46.2 20 500 23 6 45 5.8 1 Total glucose: 27.5 g/l Total yeastextract: 8.75 g/l Fermentation R11B 0.1 20 200 92.9 0 2 5.98 5.4 7 20200 82.3 0 5 5.9 5 12.4 20 300 49.5 0 5.48 21.8 20 400 18 0 40 7.12 0 2920 500 36.8 0 35 5.1 3 35.7 20 500 94.5 10 4.74 0 46.2 20 500 14.5 6 455.32 2 55 20 500 16.7 10 5.31 0.5 58.6 20 500 19.4 15 5.32 1 61.9 20 50013 15 40 5.36 2 71.7 20 500 13 15 42 5.37 0 81.1 20 500 22.9 15 5.42 2.585.6 20 500 22 15 45 5.48 1 97.5 20 500 108 15 45 6.47 0 117.7 20 500 157.38 0 Total glucose: 63 g/l Total yeast extract: 14.5 g/l

TABLE 32 Concentrations (g/l) Conversion Calc OH-can Conv.rates (g/l/h)Sample Time OH-can Canren. Total (%) (g/l) Calculated MeasuredFermentation R11A: Canrenone conversion R11A-0 0.10 0.00 5.41 5.41R11A-7 7.00 0.18 4.89 5.07 3.58 0.18 0.03 0.03 R11A- 21.80 2.02 2.124.14 48.75 2.44 0.15 0.12 22 R11A- 29.00 3.67 4.14 7.81 47.03 4.48 0.280.23 29 R11A- 35.70 6.68 1.44 8.12 82.27 7.74 0.49 0.45 36 R11A- 46.207.09 0.41 7.51 94.48 8.59 0.08 0.04 46 Fermentation R11B: Canrenoneconversion R11B-0 0.1 0.00 5.60 5.60 R11B-7 7.0 0.20 4.98 5.18 3.78 0.190.03 0.03 R11B- 21.8 2.51 2.46 4.97 50.49 2.52 0.16 0.16 22 R11B- 29.04.48 16.99 21.47 20.87 4.69 0.30 0.27 29 R11B- 35.7 8.18 10.35 18.5344.16 9.70 0.75 0.55 36 R11B- 55.0 17.03 13.20 30.23 56.33 19.50 0.320.36 55 R11B- 58.6 20.80 11.73 32.53 63.95 21.97 0.69 1.05 59 R11B- 61.922.19 8.62 30.81 72.02 24.50 0.77 0.42 62 R11B- 71.7 26.62 3.61 30.2388.06 29.46 0.51 0.45 72 R11B- 81.1 27.13 2.05 29.18 92.97 30.32 0.090.05 81 R11B- 85.6 26.87 2.02 28.88 93.02 30.11 −0.04 −0.06 86 R11B-97.5 23.95 1.71 25.66 93.34 30.22 0.01 −0.25 97 R11B- 117.7 24.10 1.6825.79 93.47 30.26 0.00 0.01 118

EXAMPLE 15

Various cultures were tested for effectiveness in the bioconversion ofcanrenone to 11α-canrenone according to the methods generally describedabove.

A working cell bank of each of Asiergillus niger ATCC 11394, Rhizopusarrhizus ATCC 11145 and Rhizopus stolonifer ATCC 6227b was prepared inthe manner described in Example 5. Growth medium (50 ml) having thecomposition set forth in Table 18 was inoculated with a suspension ofspores (1 ml) from the working cell bank and placed in an incubator. Aseed culture was prepared in the incubator by fermentation at 26° C. forabout 20 hours. The incubator was agitated at a rate of 200 rpm.

Aliquots (2 ml) of the seed culture of each microorganism were used toinoculate transformation flasks containing the growth medium (30 ml) ofTable 18. Each culture was used for inoculation of two flasks, a totalof six. Canrenone (200 mg) was dissolved in methanol (4 ml) at 36° C.,and a 0.5 ml aliquot of this solution was introduced into each of theflasks. Bioconversion was carried out generally under the conditionsdescribed in Example 5 with additions of 50% by weight glucose solution(1 ml) each day. After the first 72 hours the following observationswere made on the development of mycelia in the respective transformationfermentation flasks:

ATCC 11394—good even growth

ATCC 11145—good growth in first 48 hours, but mycelial clumped into aball; no apparent growth in last 24 hours;

ATCC 6227b—good growth; mycelial mass forming clumped ball.

Samples of the broth were taken to analyze for the extent ofbioconversion. After three days, the fermentation using ATCC 11394provided conversion to 11α-hydroxycanrenone of 80 to 90%; ATCC 11145provided a conversion of 50%; and ATCC 6227b provided a conversion of 80to 90%.

EXAMPLE 16

Using the substantially the method described in Example 15, theadditional microorganisms were tested for effectiveness in theconversion of canrenone to 11α-hydroxycanrenone. The organisms testedand the results of the tests are set forth in Table 33:

TABLE 33 Cultures tested for Bioconversion of canrenone to 11alpha-hydroxy-canrenone re- approximate Culture ATTC# media¹ sultsconversion Rhizopus oryzae  1145 CSL + 50%  — Rhizopus stolonifer 6227bCSL + 80-90% — Aspergillus nidulans 11267 CSL + 50%  80%  Aspergillusniger 11394 CSL + 80-90% — Aspergillus ochraceus NRRL CSL + 90%  405Aspergillus ochraceus 18500 CSL + 90%  Bacillus subtilis 31028 P&CSL −0% 0% Bacillus subtilis 31028 CSL − 0% 0% Bacillus sp. 31029 P&CSL − 0%0% Bacillus sp. 31029 CSL − 0% * Bacillus megaterium 14945 P&CSL + 5%80%* Bacillus megaterium 14945 CSL + 5% 10%* Trichothecium roseum 12519CSL + 80%* 90%* Trichothecium roseum  8685 CSL + 80%* 90%* Streptomycesfradiae 10745 CSL + <5%  <10%  Streptomyces fradiae 10745 TSB − * *Streptomyces 13664 CSL − 0% * lavendulae Streptomyces 13664 TSB − 0% 0%lavendulae Nocardiodes simplex  6946 BP − 0% 0% Nocardiodes simplex13260 BP − * * Pseudomonas sp. 14696 BP − * * Pseudomonas sp. 14696CSL + <5%  <10%  Pseudomonas sp. 14696 TSB − 0% * Pseudomonas sp. 13261BP + * <10%  Pseudomonas cruciviae 13262 BP # <10%  Pseudomonas putida15175 BP − 0% 0% *formation of other unidentified products ¹Media:CSL-corn steep liquor; TSB-tryptic soy broth; P&CSL-peptone and acornsteep liquor; BP-beef extract and peptone.

EXAMPLE 17

Various microorganisms were tested for effectiveness in the conversionof canrenone to 9α-hydroxycanrenone. Fermentation media for the runs ofthis Example were prepared as set forth in Table 34:

TABLE 34 Soybean Meal: dextrose 20 g soybean meal 5 g NaCl 5 g yeastextract 5 g KH₂PO₄ 5 g water to 1 L pH 7.0 Peptone/yeastextract/glucose: glucose 40 g bactopeptone 10 g yeast extract 5 g waterto 1 L Mueller-Hinton: beef infusion 300 g casamino acids 17.5 g starch1.5 g water to 1 L

Fungi were grown in soybean meal medium and in peptone-yeast extractglucose; atinomycetes and eubacteria were grown in soybean meal (plus0.9% by weight Na formate for biotransformations) and in Mueller-Hintonbroth.

Starter cultures were inoculated with frozen spore stocks (20 ml soybeanmeal in 250 ml Erlenmayer flask). The flasks were covered with a milkfilter and bioshield. Starter cultures (24 or 48 hours old) were used toinoculate metabolism cultures (also 20 ml in 250 ml Erlenmeyerflask)—with a 10% to 15% crossing volume—and the latter incubated for 24to 48 hours before addition of steroid substrate for the transformationreaction.

Canrenone was dissolved/suspended in methanol (20 mg/ml), filtersterilized, and added to the cultures to a final concentration of 0.1mg/ml. All transformation fermentation flasks were shaken at 250 rpm(21″ throw) in a controlled temperature room at 26° C. and 60% humidity.

Biotransformations were harvested at 5 and 48 hours, or at 24 hours,after addition of substrate. Harvesting began with the addition of ethylacetate (23 ml) or methylene chloride to the fermentation flask. Theflasks were then shaken for two minutes and the contents of each flaskpoured into a 50 ml conical tube. To separate the phases, tubes werecentrifuged at 4000 rpm for 20 minutes in a room temperature unit. Theorganic layer from each tube was transferred to a 20 ml borosilicateglass vial and evaporated in a speed vac. Vials were capped and storedat −20° C.

To obtain material for structure determination, biotransformations werescaled up to 500 ml by increasing the number of shake flaskfermentations to 25. At the time of harvest (24 or 48 hours afteraddition of substrate), ethyl acetate was added to each flaskindividually, and the flasks were capped and put back on the shaker for20 minutes. The contents of the flasks were then poured intopolypropylene bottles and centrifuged to separate the phases, or into aseparatory funnel in which phases were allowed to separate by gravity.The organic phase was dried, yielding crude extract of steroidscontained in the reaction mixture.

Reaction product was analyzed first by thin layer chromatography onsilica gel (250 μm) fluorescence backed plates (254 nm). Ethyl acetate(500 μL was added to each vial containing dried ethyl acetate extractfrom the reaction mixture.Further analyses were conducted by highperformance liquid chromatography and mass spectrometry. Plates weredeveloped in a 95:5 v/v chloroform/methanol medium.

Further analysis was conducted by high performance liquid chromatographyand mass spectrometry. A waters HPLC with Millennium software,photodiode array detector and autosampler was used. Reversed phase HPLCused a waters NovaPak C-18 (4 μm particle size) RadialPak 4 mmcartridge.The 25 minute linear solvent gradient began with the column initializedin water:acetonitrile (75:25), and ended at water:acetonitrile (25:75).This was followed by a three minute gradient to 100% acetonitrile and 4minutes of isocratic wash before column regeneration in initialconditions.

For LC/MS, ammonium acetate was added to both the acetonitrile and waterphases at a concentration of 2 nM. Chromatography was not significantlyaffected. Eluant from the column was split 22:1, with the majority ofthe material directed to the PDA detector. The remaining 4.5% of thematerial was directed to the electrospray ionizing chamber of an SciexAPI III mass spectrometer. Mass spectrometry was accomplished inpositive mode. An analog data line from the PDA detector on the HPLCtransferred a single wave length chromatogram to the mass spectrometerfor coanalysis of the UV and MS data.

Mass spectrometric fragmentation patterns proved useful in sorting fromamong the hydroxylated substrates. The two expected hydroxylatedcanrenones, 11α-hydroxy- and 9α-hydroxy, lost water at differentfrequencies in a consistent manner which could be used as a diagnostic.Also, the 9α-hydroxycanrenone formed an ammonium adduct more readilythan did 11α-hydroxycanrenone. Set forth in Table 35 is a summary of theTLC, HPLC/UV and LC/MS data for canrenone fermentations, showing whichof the tested microorganism were effective in the bioconversion ofcanrenone to 9α-hydroxycanrenone. Of these, the preferred microorganismwas Corynespora cassiicola ATCC 16718.

TABLE 35 Summary of TLC, HPLC/UV, and LC/MS Data for CanrenoneFermentations Evidence for 9α-OH-canrenone MS: 357 TLC HPLC-peak (M +H), spot at at 9αOH- 339(—H₂O) 9α-QH- canrenone & 375 Culture AD w/UV(+NH₄) Absidia coerula ATCC n y y/n 6647 Absidia glauca ATCC n 22752Actinomucor elegans ATCC tr y tr 6476 Aspergillus flavipes tr ATCC 1030Aspergillus fumigatus tr y n ATCC 26934 Aspergillus nidulans tr y y ATCC11267 Aspergillus niger ATCC n y y 16888 Aspergillus niger ATCC n y n26693 Aspergillus ochraceus n y n ATCC 18500 Bacterium cyclo-oxydans ntr n (Searle) ATCC 12673 Beauveria bassiana ATCC tr y y 7159 Beauveriabassiana ATCC y y y 13144 Botryosphaeria obtusa y tr tr IMI 038560Calonectria decora ATCC n tr y 14767 Chaetomium cochliodes tr tr y/nATCC 10195 Comomonas testasteroni tr tr n (Searle) ATCC 11996Corynespora cassiicola y y y ATCC 16718 Cunninghamella y y y blakesleanaATCC 8688a Cunninghamella y y y echinulata ATCC 3655 Cunninghamellaelegans y y y ATCC 9245 Curcularia clavata ATCC n y y/n 22921 Curvularialunata ATCC y n n 12071 Cylindrocarpon tr n n radicicola (Searle) ATCC11011 Epicoccum humucola ATCC y y y 12722 Epicoccum oryzae ATCC tr tr tr12724 Fusarium oxysporum ATCC tr 7601 Fusarium oxysporum f. sp. n cepaeATCC 11171 Gibberella fujikuroi tr y y ATCC 14842 Gliocladiumdeliquescens y tr tr ATCC 10097 Gongronella butieri ATCC y y UV? y 22822Hypomyces chrysospermus y y y Tul. IMI 109891 Lipomyces lipofer ATCC n10792 Melanospora ornata ATCC tr n n 26180 Mortierella isabellinay y y nATCC 42613 Mucor grisco-cyanus ATCC n 1207a Mucor mucedo ATCC 4605 tr yy Mycobacterium fortuitumn ATCC 6842 Myrothecium verrucaria tr tr y ATCC9095 Nocardia aurentia n tr n (Searle) ATCC 12674 Nocardia cancicruria yy n (Searle) Nocardia corallina ATCC n 19070 Paecilomyces carneus n y nATCC 46579 Penicillium chrysogenum n ATCC 9480 Penicillium patulum ATCCy y y/n 24550 Penicillium purpurogenum tr y y ATCC 46581 Pithomycesatro- tr y tr olivaceus ATCC 6651 Pithomyces cynodontis n tr tr ATCC26150 Phycomyces blakesleeanus y y y/n Pycnosporium sp. ATCC y y y/n12231 Rhizopogon sp. Rhizopus arrhizus ATCC tr y n 11145 Rhizopusstolonifer ATCC n 6227b Rhodococcus equi ATCC n tr n 14887 Rhodococcusequi ATCC tr tr n 21329 Rhodococcus sp. n n n Rhodococcus rhodochrous ntr n ATCC 19150 Saccharopolyspora y y y erythaea ATCC 11635 Sepedoniumampullosporum n n n IMI 203033 Sepedonium chrysospermum n ATCC 13378Septomyxa affinis ATCC n y UV? y/n 6737 Stachylidium bicolor y y y/nATCC 12672 Streptomyces n californicus ATCC 15436 Streptomyces ncinereocrocatus ATCC 3443 Streptomyces coelicolor n ATCC 10147Streptomyces flocculus ATCC 25453 Streptomyces fradiae n ATCC 10745Streptomyces griseus n subsp. ariseus ATCC 13968 Streptomyces griseus nATCC 11984 Streptomyces hydrogenans n ATCC 19631 Streptomyces y y yhygroscopicus ATCC 27438 Streptomyces lavendulae n Panlab 105Streptomyces n paucisporogenes ATCC 25489 Streptomyces n tr trpurpurascens ATCC 25489 Streptomyces roseochromogenes ATCC 13400Streptomyces spectabilis n ATCC 27465 Stysanus microsporus ATCC 2833Syncephalastrum n racemosum ATCC 18192 Thamnidium elegans ATCC 18191Thamnostylum piriforme y tr y ATCC 8992 Thielavia terricolan n ATCC13807 Trichoderma viride ATCC n 26802 Trichothecium roseum tr y y/n ATCC12543 Verticillium theobromae y tr tr ATCC 12474

EXAMPLE 18

Various cultures were tested for effectiveness in the bioconversion ofandrostendione to 11α-hydroxyandrostendione according to the methodsgenerally described above.

A working cell bank of each of Aspergillus ochraceus NRRL 405 (ATCC18500); Asnergillus niger ATCC 11394; Asperaillus nidulans ATCC 11267;Rhizopus oryzae ATCC 11145; Rhizopus stolonifer ATCC 6227b;Trichothecium roseum ATCC 12519 and ATCC 8685 was prepared essentiallyin the manner described in Example 4. Growth medium (50 ml) having thecomposition set forth in Table 18 was inoculated with a suspension ofspores (1 ml) from the working cell bank and placed in an incubator. Aseed culture was prepared in the incubator by fermentation at 26° C. forabout 20 hours. The incubator was agitated at a rate of 200 rpm.

Aliquots (2 ml) of the seed culture of each microorganism were used toinoculate transformation flasks containing the growth medium (30 ml) ofTable 15. Each culture was used for inoculation of two flasks, a totalof 16. Androstendione (300 mg) was dissolved in methanol (6 ml) at 36°C., and a 0.5 ml aliquot of this solution was introduced into each ofthe flasks. Bioconversion was carried out generally under the conditionsdescribed in Example 6 for 48 hours. After 48 hours samples of the brothwere pooled and extracted with ethyl acetate as in Example 17. The ethylacetate was concentrated by evaporation, and samples were analyzed bythin layer chromatography to determine whether a product having achromatographic mobility similar to that of 11α-hydroxy-androstendionestandard (Sigma Chemical Co., St. Louis) was present. The results areshown in Table 36. Positive results are indicated as “+”.

TABLE 36 Bioconversion of androstendione to 11 alpha-hydroxy-androstendione TLC Culture ATTC # media results Rhizopus oryzae11145 CSL + Rhizopus stolonifer 6227b CSL + Aspergillus nidulans 11267CSL + Aspergillus niger 11394 CSL + Aspergillus ochraceus NRRL 405 CSL +Aspergillus ochraceus 18500 CSL + Trichothecium roseum 12519 CSL +Trichothecium roseum 8685 CSL +

The data in Table 36 demonstrate that each of listed cultures wascapable of producing a compound from androstendione having the same Rfvalue as that of the 11α-hydroxyandrostendione standard.

Asiergillus ochraceus NRRL 405 (ATCC 18500) was retested by the sameprocedure described above, and the culture products were isolated andpurified by normal phase silica gel column chromatography using methanolas the solvent. Fractions were analyzed by thin layer chromatography.TLC plates were Whatman K6F silica gel 60 Å, 10×20 size, 250μ thickness.The solvent system was methanol:chloroform, 5:95, v/v. The crystallizedproduct and 11α-hydroxyandrostendione standard were both analyzed byLC-MS and NMR spectroscopy. Both compounds yielded similar profiles andmolecular weights.

EXAMPLE 19

Various microorganisms were tested for effectiveness in the conversionof mexrenone to 11β-hydroxymexrenone. Fermentation media for thisexample were prepared as described in Table 34.

The fermentation conditions and analytical methods were the same asthose in Example 17. TLC plates and the solvent system were as describedin Example 18. The rationale for chromatographic analysis is as follows:11α-hydroxymexrenone and 11α-hydroxycanrenone have the samechromatographic mobility. 11α-hydroxycanrenone and 9α-hydroxycanrenoneexhibit the same mobility pattern as 11α-hydroxyandrostendione and11β-hydroxyandrostendione. Therefore, 11β-hydroxymexrenone should havethe same mobility as 9α-hydroxycanrenone. Therefore, compounds extractedfrom the growth media were run against 9α-hydroxycanrenone as astandard. The results are shown in Table 36.

TABLE 37 Summary of TLC Data for 11β-hydroxymexrenone Formation fromMexrenone Spot Microorganism Medium¹ Character² Absidia coerula ATCC6647 M, S strong Aspergillus niger ATCC S, P faint (S) 16888 ? (P)Beauveria bassiana ATCC P strong 7159 Beauveria bassiana ATCC S, P ?, ?13144 Botryosphaeria obtusa IMI faint 038560 Cunninghamella blakesleeanaATCC 8688a S, P strong echinulata ATCC 3655 S, P strong elegans ATCC9245 S, P strong Curvularia lunata ATCC S strong 12017 Gongronellabutleri ATCC S, P strong 22822 Penicillium patulum ATCC S, P strong24550 Penicillium purpurogenum S, P strong ATCC 46581 Pithomycesatro-olivaceus S, P faint IFO 6651 Rhodococcus equi ATCC M faint 14887Saccharopolyspora erythaea M, SF faint ATCC 11635 Streptomyceshygroscopicus M, SF strong ATCC 27438 Streptomyces purpurascens M, SFfaint ATCC 25489 Thamnidium elegans ATCC S, P faint 18191 Thamnostylumpiriforme S, P faint ATCC 8992 Trichothecium roseum ATCC P, S faint (P)12543 ? (S) ¹M = Mueller-Hinton P = PYG (peptone/yeast extract/glucose)S = soybean meal SF = soybean meal plus formate ²? = questionabledifference from no substrate control

These data suggest that the majority of the organisms listed in thistable produce a product similar or identical to 11β-hydroxymexrenonefrom mexrenone.

EXAMPLE 20

Scheme 1: Step 1: Preparation of5′R(5′α),7′β-20′-Aminohexadecahydro-11′β-hydroxy-10′a,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile.

Into a 50 gallon glass-line reactor was charged 61.2 L (57.8 kg) of DMFfollowed by 23.5 Kg of 11-hydroxycanrenone 1 with stirring. To themixture was added 7.1 kg of lithium chloride. The mixture was stirredfor 20 minutes and 16.9 kg of acetone cyanohydrin was charged followedby 5.1 kg of triethylamine. The mixture was heated to 85° C. andmaintained at this temperature for 13-18 hours. After the.reaction 353 Lof water was added followed by 5.6 kg of sodium bicarbonate. The mixturewas cooled to 0° C., transferred to a 200 gallon glass-lined reactorwith quenched with 130 kg of 6.7% sodium hypochlorite solution slowly.The product was filtered and washed with 3×40 L portions or water togive 21.4 kg of the product. enamine.

EXAMPLE 21

Scheme 1: Step 2: Preparation of4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrile.

Into a 200 gallon glass-lined reactor was charged 50 kg of enamine 2,approximately 445 L of 0.8 N dilute hydrochloric acid and 75 L ofmethanol. The mixture was heated to 80° C. for 5 hours, cooled to 0° C.for 2 hours. The solid product was filtered to give 36.5 kg of dryproduct diketone.

EXAMPLE 22

Scheme 1: Step 3A: Preparation of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

A 4-neck 5-L bottom flask was equipped with mechanical stirrer, pressureequalizing addition funnel with nitrogen inlet tube, thermometer andcondenser with bubbler. The bubbler was connected via tygon tubing totwo 2-L traps, the first of which was empty and placed to preventback-suction of the material in the second trap (1 L of concentratedsodium hypochlorite solution) into the reaction vessel. The diketone 3(79.50 g; [weight not corrected for purity, which was 85%]) was added tothe flask in 3 L methanol. A 25% methanolic sodium methoxide solution(64.83 g) was placed in the funnel and added dropwise, with stirringunder nitrogen, over a 10 minute period. After the addition wascomplete, the orangish yellow reaction mixture was heated to reflux forhours. After this period, 167 mL of 4 N HCl was added (Caution: HCNevolution at this point!) dropwise through the addition funnel to thestill refluxing reaction mixture. The reaction mixture lightened incolor to a pale golden orange. The condenser was then replaced with atake-off head and 1.5 L of methanol was removed by distillation while1.5 L of water was simultaneously added to the flask through the funnel,in concert with the distillation rate. The reaction mixture was cooledto ambient temperature and extracted twice with 2.25 L were washedsuccessively with 750 mL aliquots of cold saturated NaCl solution, 1NNaOH and again with saturated NaCl. The organic layer was dried oversodium sulfate overnight, filtered and reduced in volume to ˜250 mL invacuo. Toluene (300 mL) was added and the remaining methylene chloridewas stripped under reduced pressure, during which time the product beganto form on the walls of the flask as a white solid. The contents of theflask were cooled overnight and the solid was removed by filtration. Itwas washed with 250 mL toluene and twice with 250 mL aliquots of etherand dried on a vacuum funnel to give 58.49 g of white solid was 97.3%pure by HPLC. On concentrating the mother liquor, an additional 6.76 gof 77.1% pure product was obtained. The total yield, adjusted forpurity, was 78%.

EXAMPLE 23

Scheme 1: Step 3B: Conversion of Methyl Hydrogen11α,17α-Dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone toMethyl Hydrogen17α-Hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-Lactone.

A 5-L four neck flask was equipped as in the above example, except thatno trapping system was installed beyond the bubbler. A quantity of138.70 g of the hydroxyester was added to the flask, followed by 1425 mLmethylene chloride, with stirring under nitrogen. The reaction mixturewas cooled to −5° C. using a salt/ice bath. Methanesulfonyl chloride(51.15 g, 0.447 mole) was added rapidly, followed by the slow dropwiseaddition of triethylamine (54.37 g) in 225 mL methylene chloride.Addition, which required ˜30 minutes, was adjusted so that thetemperature of the reaction never rose about 5° C. Stirring wascontinued for 1 hour post-addition, and the reaction contents weretransferred to a 12-L separatory funnel, to which was added 2100 mLmethylene chloride. The solution was washed successively with 700 mLaliquots each of cold 1N HCl, 1N NaOH, and saturated aqueous NaClsolution. The aqueous washes were combined and back-extracted with 3500mL methylene chloride. All of the organic washes were combined in a 9-Ljug, to which was added 500 g neutral alumina, activity grade II, and500 g anhydrous sodium sulfate. The contents of the jug were mixed wellfor 30 minutes and filtered. The filtrate was taken to dryness in vacuoto give a gummy yellow foam. This was dissolved in 350 mL methylenechloride and 1800 mL ether was added dropwise with stirring. The rate ofaddition was adjusted so that about one-half of the ether was added over30 minutes. After about 750 mL had been added, the product began toseparate as a crystalline solid. The remaining ether was added in 10minutes. The solid was removed by filtration, and the filter cake waswashed with 2 L of ether and dried in a vacuum oven at 50° C. overnight,to give 144.61 g (88%) nearly white solid, m.p. 149°-150° C. Materialprepared in this fashion is typically 98-99% pure by HPLC (area %). Inone run, material having a melting point of 153°-153.5° C. was obtained,with a purity, as determined by HPLC area, of 99.5%.

EXAMPLE 24

Scheme 1: Step 3C: Method A: Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone.

A 1-L four neck flask was equipped as in the second example. Formic acid(250 mL) and acetic anhydride (62 mL) were added to the flask withstirring under nitrogen. Potassium formate (6.17 g) was added and thereaction mixture was heated with an oil bath to an internal temperatureof 40° C. (this was later repeated at 70° C. with better results) for 16hours. After 16 hours, the mesylate 5 was added and the internaltemperature was increased to 100° C. Heating and stirring were continuedfor 2 hours, after which the solvent was removed in vacuo on a rotavap.The residue was stirred with 500 mL ice water for fifteen minutes, thenextracted twice with 500 mL aliquots of ethyl acetate. The organicphases were combined and washed successively with cold 250 mL aliquotsof saturated sodium chloride solution (two times), 1 N sodium hydroxidesolution, and again with saturated sodium chloride. The organic phasewas then dried over sodium sulfate, filtered and taken to dryness invacuo to give a yellowish white foam, which pulverized to a glass whentouched with a spatula. The powder that formed, 14.65 g analyzed as amixture of 82.1% 6 7.4% 8 and 5.7% 9 (by HPLC area %).

EXAMPLE 25

Scheme 1: Step 3C: Method B: Preparation of Methyl Hydrogen17α-Hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-Lactone.

A 5-L four neck flask was equipped as in the above example and 228.26 gacetic acid and 41.37 g sodium acetate were added with stirring undernitrogen. Using an oil bath, the mixture was heated to an internaltemperature of 100° C. The mesylate (123.65 g) was added, and heatingwas continued for thirty minutes. At the end of this period, heating wasstopped and 200 mL of ice water was added. The temperature dropped to40° C. and stirring was continued for 1 hour, after which the reactionmixture was poured slowly into 1.5 L of cold water in a 5-L stirredflask. The product separated as a gummy oil. The oil was dissolved in 1L ethyl acetate and washed with 1 L each cold saturated sodium chloridesolution, 1 N sodium hydroxide, and finally saturated sodium chlorideagain. The organic phase was dried over sodium sulfate and filtered. Thefiltrate was taken to dryness in vacuo to give a foam which collapsed toa gummy oil. This was triturated with ether for some time and eventuallysolidified. The solid was filtered and washed with more ether to afford79.59 g of a yellow white solid. This consisted of 70.4% of the desiredΔ^(9,11) enester 6, 12.3% of the Δ^(11,12) enester 8, 10.8% of the7α,9-α-lactone 9 and 5.7% unreacted 5.

EXAMPLE 26

Scheme 1: Step 3D: Synthesis of Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

A 4-neck jacketed 500 mL reactor was equipped with mechanical stirrer,condenser/bubbler, thermometer and addition funnel with nitrogen inlettube. The reactor was charged with 8.32 g of the crude enester in 83 mLmethylene chloride, with stirring under nitrogen. To this was added 4.02g dibasic potassium phosphate, followed by 12 mL oftrichloroacetonitrile. External cooling water was run through thereactor jacket and the reaction mixture was cooled to 8° C. To theaddition funnel 36 mL of 30% hydrogen peroxide was added over a 10minute period. The initially pale yellow colored reaction mixture turnedalmost colorless after the addition was complete. The reaction mixtureremained at 9±1° C. throughout the addition and on continued stirringovernight (23 hours total). Methylene chloride (150 mL) was added to thereaction mixture and the entire contents were added to ˜250 mL icewater. This was extracted three times with 150 mL aliquots of methylenechloride. The combined methylene chloride extracts were washed with 400mL cold 3% sodium sulfite solution to decompose any residual peroxide.This was followed by a 330 mL cold 1 N sodium hydroxide wash, a 400 mLcold 1 N hydrochloric acid wash, and finally a wash with 400 mL brine.The organic phase was dried over magnesium sulfate, filtered, and thefilter cake was washed with 80 mL methylene chloride. Solvent wasremoved in vacuo to give 9.10 g crude product as a pale yellow solid.This was recrystallized from ˜25 mL 2-butanone to give 5.52 g nearlywhite crystals. A final recrystallization from acetone (˜50 mL gave 3.16g long, acicular crystals, mp 241-243° C.

EXAMPLE 27

Scheme 1: Step 3: Option 1: From4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrileto Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactone.

Diketone (20 g) was charged into a clean and dried reactor followed bythe addition of 820 ml of MeOH and 17.6 ml of 25% NaOMe/MeOH solution.The reaction mixture was heated to reflux condition (˜67° C.) for 16-20hours. The product was quenched with 40 mL of 4N HCl. The solvent wasremoved at atmospheric pressure by distillation. 100 mL of toluene wasadded and the residual methanol was removed by azeotrope distillationwith toluene. After concentration, the crude hydroxyester 4 wasdissolved in 206 mL of methylene chloride and cooled to 0° C.Methanesulfonyl chloride (5 mL) was added followed by a slow addition of10.8 ml of triethylamine. The product was stirred for 45 minutes. Thesolvent was removed by vacuum distillation to give the crude mesylate 5.

In a separate dried reactor was added 5.93 g of potassium formate, 240mL of formic acid and followed by 118 mL of acetic anhydride. Themixture was heated to 70° C. for 4 hours.

The formic acid mixture was added to the concentrated mesylate solution5 prepared above. The mixture was heated to 95-105° C. for 2 hours. Theproduct mixture was cooled to 50° C. and the volatile components wereremoved by vacuum distillations at 50° C. The product was partitionedbetween 275 ml of ethyl acetate and 275 ml of water. The aqueous layerwas back extracted with 137 ml of ethyl acetate, washed with 240 ml ofcold 1N sodium-hydroxide solution and then 120 ml of saturated NaCl.After phase separation, the organic layer was concentrated to undervacuum distillation to give crude enester.

The product was dissolved in 180 mL of methylene chloride and cooled to0 to 15° C. 8.68 g of dipotassium hydrogen phosphate was added followedby 2.9 mL of trichloroacetonitrile. A 78 mL solution of 30% hydrogenperoxide was added to the mixture over a 3 minute period. The reactionmixture was stirred at 0-15° C. for 6-24 hours. After the reaction, thetwo phase mixture was separated. The organic layer was washed with 126mL of 3% sodium sulfite solution, 126 mL of 0.5 N sodium hydroxidesolution, 126 mL of 1 N hydrochloric acid and 126 mL of 10% brine. Theproduct was dried over anhydrous magnesium sulfate or filtered overCelite and the solvent methylene chloride was removed by distillation atatmospheric pressure. The product was crystallized from methylethylketone twice to give 7.2 g of eplerenone.

EXAMPLE 28

Scheme 1: Step 3: Option 2: Conversion o1′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrileto Methyl Hydrogen9,11α-Epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate, γ-Lactonewithout intermediate.

A 4-neck 5-L round bottom flask was equipped with mechanical stirrer,addition funnel with nitrogen inlet tube, thermometer and condenser withbubbler attached to a sodium hypochlorite scrubber. The diketone (83.20g) was added to the flask in 3.05 L methanol. The addition funnel wascharged with 67.85 g of a 25% (w:w) is solution of sodium methoxide inmethanol. With stirring under nitrogen, the methoxide was added dropwiseto the flask over a 15 minute period. A dark orange/yellow slurrydeveloped. The reaction mixture was heated to reflux for 20 hours and175 mL 4 N hydrochloric acid was added dropwise while refluxingcontinued. (Caution, HCN evolution during this operation!) The refluxcondenser was replaced with a takeoff head and 1.6 L of methanol wasremoved by distillation while 1.6 L of aqueous 10% sodium chloridesolution was added dropwise through the funnel, at a rate to match thedistillation rate. The reaction mixture was cooled to ambienttemperature and extracted twice with 2.25 L of aliquots of methylenechloride. The combined extracts were washed with cold 750 mL aliquots of1 N sodium hydroxide and saturated sodium chloride solution. The organiclayer was dried by azeotropic distillation of the methanol at oneatmosphere, to a final volume of 1 L (0.5% of the total was removed foranalysis).

The concentrated organic solution (hydroxyester) was added back to theoriginal reaction flask equipped as before, but without the HCN trap.The flask was cooled to 0° C. and 30.7 g methanesulfonyl chloride wasadded with stirring under nitrogen. The addition funnel was charged with32.65 g triethylamine, which was added dropwise over a 15 minute period,keeping the temperature at 5° C. Stirring was continued for 2 hours,while the reaction mixture warmed to ambient. A column consisting of 250g Dowex 50 W×8-100 acid ion exchange resin was prepared and was washedbefore using with 250 mL water, 250 mL methanol and 500 mL methylenechloride. The reaction mixture was run down this column and collected. Afresh column was prepared and the above process was repeated. A third250 g column, consisting of Dowex 1×8-200 basic ion exchange resin wasprepared and pretreated as in the acid resin treatment described above.The reaction mixture was run down this column and collected. A fourthcolumn of the basic resin was prepared and the reaction mixture againwas run down the column and collected. Each column pass was followed bytwo 250 mL methylene chloride washes down the column, and each passrequired ˜10 minutes. The solvent washes were combined with the reactionmixture and the volume was reduced in vacuo to ˜500 mL and 2% of thiswas removed for qc. The remainder was further reduced to a final volumeof 150 mL (crude mesylate solution).

To the original 5-L reaction set-up was added 960 mL formic acid, 472 mLacetic anhydride and 23.70 g potassium formate. This mixture was heatedwith stirring under nitrogen to 70° C. for 16 hours. The temperature wasthen increased to 100° C. and the crude mesylate solution was added overa thirty minute period via the addition funnel. The temperature droppedto 85° C. as methylene chloride was distilling out of the reactionmixture. After all of it had been removed, the temperature climbed backto 100° C., and was held there for 2.5 hours. The reaction mixture wascooled to 40° C. and the formic acid was removed under pressure untilthe minimum stir volume had been reached (˜150 mL). The residue wascooled to ambient and 375 mL methylene chloride was added. The dilutedresidue was washed with cold 1 L portions of saturated sodium chloridesolution, 1 N sodium carbonate, and again with sodium chloride solution.The organic phase was dried over magnesium sulfate (150 g), and filteredto give a dark reddish brown solution (crude enester solution).

A 4-neck jacketed 1 L reactor was equipped with mechanical stirrer,condenser/bubbler, thermometer and addition funnel with nitrogen inlettube. The reactor was charged with the crude enester solution (estimated60 g) in 600 mL methylene chloride, with stirring under nitrogen. Tothis was added 24.0 g dibasic potassium phosphate, followed by 87 mLtrichloroacetonitrile. External cooling water was run through thereactor jacket and the reaction mixture was cooled to 10° C. To theaddition funnel 147 mL 30% hydrogen peroxide was added mixture over a 30minute period. The initially dark reddish brown colored reaction mixtureturned a pale yellow after the addition was complete. The reactionmixture remained at 10±1° C. throughout the addition and on continuedstirring overnight (23 hours total). The phases were separated and theaqueous portion was extracted twice with 120 mL portions of methylenechloride. The combined organic phases were then washed with 210 mL 3%sodium sulfite solution was added. This was repeated a second time,after which both the organic and aqueous parts were negative forperoxide by starch/iodide test paper. The organic phase was successivelywashed with 210 mL aliquots of cold 1 N sodium hydroxide, 1 Nhydrochloric acid, and finally two washes with brine. The organic phasewas dried azeotropically to a volume of ˜100 mL, fresh solvent was added(250 mL and distilled azeotropically to the same 100 mL and theremaining solvent was removed in vacuo to give 57.05 g crude product asa gummy yellow foam. A portion (51.01 g) was further dried to a constantweight of 44.3 g and quantitatively analyzed by HPLC. It assayed at27.1% EPX.

EXAMPLE 29

11α-Hydroxyandrostendione (429.5 g) and toluene sulfonic acid hydrate(7.1) were charged to a reaction flask under nitrogen. Ethanol (2.58 L)was added to the reactor, and the resulting solution cooled to 5° C.Triethyl orthoformate (334.5 g) was added to the solution over a 15minute period at 0° to 15° C. After the triethyl orthoformate additionwas complete the reaction mixture was warmed to 40° C. and reacted atthat temperature for 2 hours, after which the temperature was increasedto reflux and reaction continued under reflux for an additional 3 hours.The reaction mixture was cooled under vacuum and the solvent removedunder vacuum to yield 3-ethoxyandrosta-3,5-diene-17-one.

EXAMPLE 30 Formation of Enamine from 11α-Hydroxycanrenone

Sodium cyanide (1.72 g) was placed in 25 mL 3-neck flask fitted with amechanical stirrer. Water (2.1 mL) was added and the mixture was stirredwith heating until the solids dissolved. Dimethylformamide (15 mL) wasadded followed by 11α-hydroxycanrenone (5.0 g). A mixture of water (0.4mL) and sulfuric acid (1.49 g) was added to mixture. The mixture washeated to 85° C. for 2.5 hours at which time HPLC analysis showedcomplete conversion to product. The reaction mixture was cooled to roomtemperature. Sulfuric acid (0.83 g) was added and the mixture stirredfor one half hour. The reaction mixture was added to 60 mL water cooledin an ice bath. The flask was washed with 3 mL DMF and 5 mL water. Theslurry was stirred for 40 min. and filtered. The filter cake was washedtwice with 40 mL water and dried in a vacuum oven at 60° C. overnight toyield the 11α-hydroxy enamine, i.e.,5′R(5′α),7′β-20′-aminohexadecahydro-11β-hydroxy-10′α,13′α-dimethyl-3′,5-dioxospiro[furan-2(3H),17′α(5′H)-[7,4]metheno[4H]cyclopenta[a]phenanthrene]-5′-carbonitrile(4.9 g).

EXAMPLE 31 Conversion of 11α-Hydroxycanrenone to Diketone

Sodium cyanide (1.03 g) was added to a 50 mL 3-neck flask fitted with amechanical stirrer. Water (1.26 mL) was added and the flask was heatedslightly to dissolve the solid. Dimethylacetamide [or dimethyformamide](9 mL) was added followed by 11α-hydroxycanrenone (3.0 g). A mixture ofsulfuric acid (0.47 mL) and water (0.25 mL) was added to the reactionflask while stirring. The mixture was heated to 95° C. for 2 hours. HPLCanalysis indicated that the reaction was complete. Sulfuric acid (0.27mL) was added and the mixture stirred for 30 min. Additional water (25mL) and sulfuric acid (0.90 mL) were introduced and the reaction mixturestirred for 16 hours. The mixture was then cooled in an ice bath to5-10° C. The solid was isolated by filtering through a sintered glassfilter followed by washing twice with water (20 mL). The solid diketone,i.e.,4′S(4′α),7′α-Hexadecahydro-11′α-hydroxy-10′β,13′β-dimethyl-3′,5,20′-trioxospiro[furan-2(3H),17′β-[4,7]methano[17H]cyclopenta[a]phenanthrene]-5′β(2′H)-carbonitrilewas dried in a vacuum oven to yield 3.0 g of a solid.

EXAMPLE 32

A suspension of 5.0 g of the diketone produced in the manner describedin Example 31 in methanol (100 mL) was heated to reflux and a 25%solution of potassium methoxide in methanol (5.8 mL) was added over 1min. The mixture became homogeneous. After 15 min., a precipitate waspresent. The mixture was heated at reflux and again became homogeneousafter about 4 hours. Heating at reflux:was continued for a total of 23.5hours and 4.0 N HCl (10 mL) was added. A total of 60 mL of a solution ofhydrogen cyanide in methanol was removed by distillation. Water (57 mL)as added to the distillation residue over min. The temperature of thesolution was raised to 81.5° during water addition and an additional 4mL of hydrogen cyanide/methanol solution was removed by distillation.After water addition was complete, the mixture became cloudy and theheat source was removed. The mixture was stirred for 3.5 hours andproduct slowly crystallized. The suspension was filtered and thecollected solid was washed with water, dried in a stream of air on thefunnel, and dried at 92° (26 in. Hg) for 16 hours to give 2.98 g of anoff-white solid. The solid was 91.4% of the hydroxyester, i.e., methylhydrogen 11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone by weight. The yield was 56.1%.

EXAMPLE 33

Diketone prepared in the manner described in Example 31 was charger intoa cleaned and dried 3-neck reaction flask equipped with a thermometer, aDean Stark trap and a mechanical stirrer. Methanol (24 mL) was chargedto the reactor at room temperature (22° C.) and the resulting slurrystirred for 5 min. A 25% by weight solution of sodium methoxide inmethanol (52.8 mL) was charged to the reactor and the mixture stirredfor 10 min. at room temperature during which the reaction mixture turnedto a light brown clear solution and a slight exotherm was observed (2-3°C.). The addition rate was controlled to prevent the pot temperaturefrom exceeding 30° C. The mixture was thereafter heated to refluxconditions (about 67° C.) and continued under reflux for 16 hrs. Asample was then taken and analyzed by HPLC for conversion. The reactionwas continued.under reflux until the residual diketone was not greaterthan 3% of the diketone charge. During reflux 4 N HCl (120 mL) wascharged to the reaction pot resulting in the generation of HCN which wasquenched in a scrubber.

After conclusion of the reaction, 90-95% of the methanol solvent wasdistilled out of the reaction mixture at atmospheric pressure. Headtemperature during distillation varied from 67-75° C. and the distillatewhich contained HCN was treated with caustic and bleach before disposal.After removal of methanol the reaction mixture was cooled to roomtemperature, solid product beginning to precipitate as the mixturecooled in the 40-45° C. range. An aqueous solution containing optionally5% by weight sodium bicarbonate (1200 mL) at 25° C. was charged to thecooled slurry and the resultant mixture then cooled to 0° C. in about 1hr. Sodium bicarbonate treatment was effective to eliminate residualunreacted diketone from the reaction mixture. The slurry was stirred at0° C. for 2 hrs. to complete the precipitation and crystallization afterwhich the solid product was recovered by filtration and the filter cakewashed with water (100 mL). The product was dried at 80-90° C. under 26″mercury vacuum to constant weight. Water content after drying was lessthan 0.25% by weight. Adjusted molar yield was around 77-80% by weight.

EXAMPLE 34

Diketone as prepared in accordance with Example 31 (1 eq.) was reactedwith sodium methoxide (4.8 eqs.) in a methanol solvent in the presenceof zinc iodide (1 eq.). Work up of the reaction product can be either inaccordance with the extractive process described herein, or by anon-extractive process in which methylene chloride extractions, brineand caustic washes, and sodium sulfate drying steps are eliminated. Alsoin the non-extractive process, toluene was replaced with 5% by weightsodium bicarbonate solution.

EXAMPLE 35

The hydroxyester prepared as by Example 34 (1.97 g) was combined withtetrahydrofuran (20 mL) and the resulting mixture cooled to −70° C.Sulfuryl chloride (0.8 mL) was added and the mixture was stirred for 30min., after which imidazole (1.3 g) was added. The reaction mixture waswarmed to room temperature and stirred for an additional 2 hrs. Themixture was then diluted with methylene chloride and extracted withwater. The organic layer was concentrated to yield crude enester (1.97g). A small sample of the crude product was analyzed by HPLC. Theanalysis showed that the ratio of 9,11-olefin: 11,12-olefin: 7,9-lactonewas 75.5:7.2:17.3. When carried out at 0° C. but otherwise as describedabove, the reaction yielded a product in which the 9,11-olefin:11,12-olefin: 7,9-lactone distribution was 77.6:6.7:15.7. This procedurecombines into one step the introduction of a leaving group andelimination thereof for the introduction of the 9,11-olefin structure ofthe enester, i.e., reaction was sulfuryl chloride causes the 11α-hydroxygroup of the hydroxy ester of Formula V to be replaced by halide andthis is followed by dehydrohalogenation to the Δ-9,11 structure. Thusformation of the enester is effected without the use of a strong.acid(such as formic) or a drying agent such as acetic anhydride. Alsoeliminated is the refluxing step of the alternative process whichgenerates carbon monoxide.

EXAMPLE 36

Hydroxyester (20 g) prepared as by Example 34, and methylene chloride(400 mL) were added to a clean dry three-neck round bottom flask fittedwith a mechanical stirrer, addition funnel and thermocouple. Theresulting mixture was stirred at ambient temperature until completesolution was obtained. The solution was cooled to 5° C. using an icebath. Methanesulfonyl chloride (5 mL) was added to the solution ofCH₂Cl₂ containing the hydroxyester, rapidly followed by the slowdropwise addition of triethylamine (10.8 mL). The addition rate wasadjusted so that the temperature of the reaction did not exceed 5° C.The reaction was very exothermic; therefore cooling was necessary. Thereaction mixture was stirred at about 5° C. for 1 h. When the reactionwas complete (HPLC and TLC analysis), the mixture was concentrated atabout 0° C. under 26 in Hg vacuum until it became a thick slurry. Theresulting slurry was diluted with CH₂Cl₂ (160 mL), and the mixture wasconcentrated at about 0° C. under 26 in Hg vacuum to obtain aconcentrate. The purity of the concentrate (mesylate product of FormulaIV wherein R³═H and —A—A— and —B—B— are both —CH₂—CH₂—, i.e., methylhydrogen 11α,17α-dihydroxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone to methyl hydrogen17α-hydroxy-11α-(methylsulfonyl)oxy-3-oxopregn-4-ene-7α,21-dicarboxylate,γ-lactone was found to be 82% (HPLC area %). This material was used forthe next reaction without isolation.

Potassium formate (4.7 g), formic acid (16 mL) and acetic anhydride (8mL, 0.084 mol) were added to a clean dry reactor equipped withmechanical stirrer, condenser, thermocouple and heating mantle. Theresulting solution was heated to 70° C. and stirred for about 4-8 hours.The addition of acetic anhydride is exothermic and generated gas (CO),so that the rate of addition had to be adjusted to control bothtemperature and gas generation (pressure). The reaction time to preparethe active eliminating reagent was dependent on the amount of waterpresent in the reaction (formic acid and potassium formate containedabout 3-5% water each). The elimination reaction is sensitive to theamount of water present; if there is >0.1% water (KF), the level of the7,9-lactone impurity may be increased. This by product is difficult toremove from the final product. When the KF showed <0.1% water, theactive eliminating agent was transferred to the concentrate of mesylate(0.070 mol) prepared in the previous step. The resulting solution washeated to 95° C. and the volatile material was distilled off andcollected in a Dean Stark trap. When volatile material evolution ceased,the Dean Stark trap was replaced with the condenser and the reactionmixture was heated for additional 1 h at 95° C. Upon completion (TLC andHPLC analysis; <0.1% starting material) the content was cooled to 50° C.and vacuum distillation was started (26 in Hg/50° C.). The mixture wasconcentrated to a thick slurry and then cooled to ambient temperature.The resulting slurry was diluted with ethyl acetate (137 mL) and thesolution was stirred for 15 min. and diluted with water (137 mL). Thelayers were separated, and the aqueous lower layer was re-extracted withethyl acetate (70 mL). The combined ethyl acetate solution was washedonce with brine solution (120 mL) and twice with ice cold 1N NaOHsolution (120 mL each). The pH of aqueous was measured, and the organiclayer rewashed if the pH of the spent wash liquor was <8. When the pH ofthe spent wash was observed to be >8, the ethyl acetate layer was washedonce with brine solution (120 mL) and concentrated to dryness by rotaryevaporation using a 50° C. water bath. The resulting enester, solidproduct i.e., methyl hydrogen17α-hydroxy-3-oxopregna-4,9(11)-diene-7α,21-dicarboxylate, γ-lactoneweighed 92 g (77% mol yield).

EXAMPLE 37

Hydroxyester (100 g; 0.22 mol) prepared as by Example 34 was charged toa 2 L 3-neck round bottom flask equipped with mechanical stirrer,addition funnel, and thermocouple. A circulating cooling bath was usedwith automatic temperature control. The flask was dried prior toreaction because of the sensitivity of methanesulfonyl chloride towater.

Methylene chloride (1 L) was charged to the flask and the hydroxyesterdissolved therein under agitation. The solution was cooled to 0° C. andmethane sulfonyl chloride (25 mL; 0.32 mol) was charged to the flask viathe addition funnel. Triethylamine (50 mL; 0.59 mol) was charged to thereactor via the addition funnel and the funnel was rinsed withadditional methylene chloride (34 mL). Addition of triethylamine washighly exothermic. Addition time was around 10 min. under agitation andcooling. The charge mixture was cooled to 0° C. and held at thattemperature under agitation for an additional 45 min. during which thehead space of the reaction flask was flushed with nitrogen. A sample ofthe reaction mixture was then analyzed by thin layer chromatography andhigh performance liquid chromatography to check for reaction completion.The mixture was thereafter stirred at 0° C. for an additional 30 min.and checked again for reaction completion. Analysis showed the reactionto be substantially complete at this point; the solvent methylenechloride was stripped at 0° C. under 26″ mercury vacuum. Gaschromatography analysis of the distillate indicated the presence of bothmethane sulfonyl chloride and triethylamine. Methylene chloride (800 mL)was thereafter charged to the reactor and the resulting mixture wasstirred for 5 min. at a temperature in the range of 0-15° C. The solventwas again stripped at 0-5° C. under 26″ mercury vacuum yielding themesylate of Formula IV wherein R³ is H, —A—A— and —B—B— are —CH₂—CH₂—and R¹ is methoxy carbonyl. The purity of the product was about 90-95area %.

To prepare an elimination reagent, potassium formate (23.5 g; 0.28 mol),formic acid (80 mL) and acetic anhydride (40 mL) were mixed in aseparate dried reactor. Formic acid and acetic anhydride were pumpedinto the reactor and the temperature was maintained not greater than 40°C. during addition of acetic anhydride. The elimination reagent mixturewas heated to 70° C. to scavenge water from the reaction system. Thisreaction was continued until the water content was lower than 0.3% byweight as measured by Karl Fisher analysis. The elimination reagentsolution was then transferred to the reactor containing the concentratedcrude mesylate solution prepared as described above. The resultingmixture was heated to a maximum temperature of 95° C. and volatiledistillate collected until no further distillate was generated.Distillation ceased at about 90° C. After distillation was complete, thereaction mixture was stirred at 95° C. for an additional 2 hrs. andcompletion of the reaction was checked for thin layer chromatography.When the reaction was complete, the reactor.was cooled to 50° C. and theformic acid and solvent removed from the reaction mixture under 26″mercury vacuum at 50° C. The concentrate was cooled to room temperatureand thereafter ethyl acetate (688 mL) was introduced and the mixture ofethyl acetate and concentrate stirred for 15 min. At this point, a 12%brine solution (688 mL) was introduced to assist in removing watersoluble impurities from the organic phase. The phases were then allowedto settle for 20 min. The aqueous layer was transferred to anothervessel to which an additional amount of ethyl acetate (350 mL) wascharged. This back extraction of the aqueous layer was carried out for30 min. after which the phases were allowed to settle and the ethylacetate layers combined. To the combined ethyl acetate layers, saturatedsodium chloride solution (600 mL) was charged and stirring carried outfor 30 min. The phases were then allowed to settle. The aqueous layerwas removed. An additional sodium chloride (600 mL) wash was carriedout. The organic phase was separated from the second spent wash liquor.The organic phase was then washed with 1 N sodium hydroxide (600 mL)under stirring for 30 min. The phases were settled for 30 min. to removethe aqueous layer. The pH of the aqueous layer was checked and it foundto be >7. A further wash was carried out with saturated sodium chloride(600 mL) for 15 min. The organic phase was finally concentrated under26″ mercury vacuum at 50° C. and the product recovered by filtration.The final product was a foamy brown solid when dried. Further drying at45° C. under reduced pressure for 24 hrs. yielded 95.4 g of the enesterproduct which assayed at 68.8%. The molar yield was 74.4% corrected forboth the starting hydroxy ester and the final enester.

EXAMPLE 38

The procedure of Example 37 was repeated except that the multiplewashing steps were avoided by treating the reaction solution with an ionexchange resin. Basic alumina or basic silica. Conditions for treatmentwith basic silica are set forth in Table 38. Each of these treatmentswas found effective for removal of impurities without the multiplewashes of Example 44.

TABLE 38 Factor Set point Purpose of Experiment Key results Basic 2g/125 g Treating the reaction mixture The yield alumina product withbasic alumina to remove was 93% Et₃N.HCl salt and to eliminate the 1 NNaOH and 1 N HCl washes Basic 2 g/125 g Treating the reaction mixtureThe yield silica product with basic silica which is was 95% cheaper toremove Et₃N.HCl salt and eliminate 1 N NaOH and 1 N HCl washes

EXAMPLE 39

Potassium acetate (4 g) and trifluoroacetic acid (42.5 mL) were mixed ina 100 mL reactor. trifluoroacetic anhydride (9.5 mL) was added to themixture at a rate controlled to maintain temperature during additionbelow 30° C. The solution was then heated to 30° C. for 30 min. toprovide an elimination reagent useful for converting the mesylate ofFormula IV to the enester of Formula II.

The preformed TFA/TFA anhydride elimination reagent-was added to apreviously prepared solution of the mesylate of Formula IV. Theresulting mixture was heated at 40° C. for 4½ hrs., the degree ofconversion being periodically checked by TLC or HPLC. When the reactionwas complete, the mixture was transferred to 1-neck flask andconcentrated to dryness under reduced pressure at room temperature (22°C.). Ethyl acetate (137 mL) was added to the mixture to obtain completedissolution of solid phase material after which a water/brine mixture(137 mL) was added and the resulting two phase mixture stirred for 10min. The phases were then allowed to separate for 20 min. Brine strengthwas 24% by.weight. The aqueous phase was contacted with an additionalamount of ethyl acetate (68 mL) and the two phase mixture thus preparedwas stirred for 10 min. after which it was allowed to stand for 15 min.for phase separation. The ethyl acetate layers from the two extractionswere combined and washed with 24% by weight brine (120 mL), anotheraliquot of 24% by weight brine (60 mL), 1 N sodium hydroxide solution(150 mL) and another portion of brine (60 mL). After each aqueous phaseaddition, the mixture was stirred for 10 min. and allowed to stand for15 min. for separation. The resulting solution was concentrated todryness under reduced pressure at 45° C. using a water aspirator. Thesolid product (8.09 g) was analyzed by HPLC and found to include 83.4area % of the enester, 2.45 area % of the 11,12-olefin, 1.5% of the7,9-lactone, and 1.1% of unreacted mesylate.

EXAMPLE 40

The mesylate having the structure prepared per Example 23 (1.0 g),isopropenyl acetate (10 g) and p-toluenesulfonic acid (5 mg) were placedin a 50 ml flask and heated to 90° C. with stirring. After 5 hours themixture was cooled to 25° C. and concentrated in vacuo at 10 mm of Hg.The residue was dissolved in CH₂Cl₂ (20 ml) and washed with 5% aqueousNaHCO₃. The CH₂Cl₂ layer was concentrated in vacuo to give 1.47 g of atan oil. This material was recrystallized from CH₂Cl₂/Et₂O to give 0.50g of enol acetate of Formula IV(Z).

This material was added to a mixture of sodium acetate (0.12 g) andacetic acid (2.0 ml) that had been previously heated to 100° C. withstirring. After 60 minutes the mixture was cooled to 25° C. and dilutedwith CH₂Cl₂ (20 ml). The solution was washed with water (20 ml) anddried over MgSO₄. The drying agent was removed by filtration and thefiltrate was concentrated in vacuo to give 0.4 g of the desired9,11-olefin, IV(Y). The crude product contained less than 2% of the7,9-lactone impurity.

EXAMPLE 41 Thermo Elimination of Mesylate in DMSO

A mixture of 2 g of mesylate and 5 ml of DMSO in a flask was heated at80° C. for 22.4 hours. HPLC analysis of the reaction mixture indicatedno starting material was detected. To the reaction was added water (10ml) and the precipitate was extracted with methylene chloride threetimes. The combined methylene chloride layers were washed with water,dried over magnesium sulfate, and concentrated to give the enester.

EXAMPLE 42

In a 50 mL pear-shaped flask under stirring the enester of Formula IIA(1.07 g assaying 74.4% enester), trichloroacetamide (0.32 g),dipotassium hydrogen phosphate (0.70 g) as solid were mixed withmethylene chloride (15.0 mL). A clear solution was obtained. Hydrogenperoxide (30% by weight; 5.0 mL) was added via a pipet over a 1 min.period. The resulting mixture was stirred for 6 hrs. at room temperatureat which point HPLC analysis showed that the ratio of epoxymexrenone toenester in the reaction mixture was approximately 1:1. Additionaltrichloroacetamide (0.32 g) was added to the reaction mixture andreaction continued under agitation for 8 more hours after which time theremaining proportion of enester was shown to have been reduced to 10%.Additional trichloroacetamide (0.08 g) was added and the reactionmixture was allowed to stand overnight at which point only 5% ofunreacted enester remained relative to epoxymexrenone in the mixture.

EXAMPLE 43

Enester of Formula IIA (5.4 g, assaying 74.4% enester) was added to a100 mL reactor. Trichloroacetamide (4.9 g) and dipotassium hydrogenphosphate (3.5 g) both in solid form were added to the enester followedby methylene chloride (50 mL). The mixture was cooled to 15° C. and a30% hydrogen peroxide (25 g) was added over a ten min. period. Thereaction mixture was allowed to come to 20° C. and stirred at thattemperature for 6 hrs., at which point conversion was checked by HPLC.Remaining enester was determined to be less than 1% by weight.

The reaction mixture was added to water (100 mL), the phases wereallowed to separate, and the methylene chloride layer was removed.Sodium hydroxide (0.5 N; 50 mL) was added to the methylene chloridelayer. After 20 min. the phases were allowed to separate HCl (0.5 N; 50mL) was added to the methylene chloride layer after which the phaseswere allowed to separate and the organic phase was washed with saturatedbrine (50 mL). The methylene chloride layer was dried over anhydrousmagnesium sulfate and the solvent removed. A white solid (5.7 g) wasobtained. The aqueous sodium hydroxide layer was acidified and extractedand the extract worked up to yield an additional 0.2 g of product. Yieldof epoxymexrenone was 90.2%.

EXAMPLE 44

Enester of Formula IIA was converted to epoxymexrenone in the mannerdescribed in Example 43 with the following differences: the initialcharge comprised of enester (5.4 g assaying 74.4% enester),trichloroacetamide (3.3 g), and dipotassium hydrogen phosphate (3.5 g).Hydrogen peroxide solution (12.5 mL) was added. The reaction wasconducted overnight at 20° C. after which HPLC showed a 90% conversionof enester to epoxymexrenone. Additional trichloroacetamide (3.3 g) and30% hydrogen peroxide (5.0 mL) was added and the reaction carried outfor an additional 6 hrs. at which point the residual enester was only 2%based on the enester charge. After work up as described in Example 43,5.71 g of epoxymexrenone resulted.

EXAMPLE 45

The enester of Formula IIA was converted to epoxymexrenone in the mannergenerally described in Example 43. In the reaction of this Example,enester charge was 5.4 g (assaying 74.4% enester), thetrichloroacetamide charge was 4.9 g, hydrogen peroxide charge was 25 g,dipotassium hydrogen phosphate charge was 3.5 g. The reaction was run at20° C. for 18 hrs. The residual enester was less than 2%. After work up,5.71 g of epoxymexrenone resulted.

EXAMPLE 46

Enester of Formula IIA was converted to epoxymexrenone in the mannerdescribed in Example 43 except that the reaction temperature in thisExample was 28° C. The materials charged in the reactor included enester(2.7 g), trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7g), hydrogen peroxide (17.0 g) and methylene chloride (50 mL). After 4hrs. reaction, unreacted enester was only 2% based on the enestercharge. After work up as described in Example 43, 3.0 g ofepoxymexrenone was obtained.

EXAMPLE 47

Enester of Formula IIA (17 g assaying 72% enester) was dissolved inmethylene chloride (150 mL) after which trichloroacetamide (14.9 g) wasadded under slow agitation. The temperature of the mixture was adjustedto 25° C. and the solution of dipotassium hydrogen phosphate (10.6 g) inwater (10.6 mL) was stirred into the enester substrate solution under400 rpm agitation. Hydrogen peroxide (30% by weight solution; 69.4 mL)was added to the substrate/phosphate/trichloroacetamide solution over a3-5 min. period. No exotherm or oxygen evolution was observed. Thereaction mixture thus prepared was stirred at 400 rpm and 25° C. for18.5 hrs. No oxygen evolution was observed throughout the course of thereaction. The reaction mixture was diluted with water (69.4 mL) and themixture stirred at about 250 rpm for 15 min. No temperature control wasnecessary for this operation and it was conducted essentially at roomtemperature (any temperature in the range of 5-25° C. being acceptable).The aqueous and organic layers were allowed to separate and the lowermethylene chloride layer was removed.

The aqueous layer was back extracted with methylene chloride (69.4 mL)for 15 min. under agitation of 250 rpm. The layers were allowed toseparate and the lower methylene chloride layer was removed. The aqueouslayer (177 g; pH=7) was submitted for hydrogen peroxide determination.The result (12.2%) indicating that only 0.0434 mol of hydrogen peroxidewere consumed in the reaction was 0.0307 mol of olefin. Back extractionwith a small amount of methylene chloride volume was sufficient toinsure no loss of epoxymexrenone in the aqueous layer. This result wasconfirmed with the application of a second large methylene chlorideextraction in which only trichloroacetamide was recovered.

The combined methylene chloride solutions from the above describedextractions were combined and washed with 3% by weight sodium sulfitesolution (122 mL) for at least 15 min. at about 250 rpm. A negativestarch iodide test (KI paper; no color observed; in a positive test apurple coloration indicates the presence of peroxide) was observed atthe end of the stir period.

The aqueous and organic layers were allowed to separate and the lowermethylene chloride layer removed. The aqueous layer (pH=6) wasdiscarded. Note that addition of sodium sulfite solution can cause aslight exotherm so that such addition should be carried out undertemperature control.

The methylene chloride phase was washed with 0.5 N sodium hydroxide (61mL) for 45 min. at about 250 rpm and a temperature in the range of15-25° C. (pH=12-13). Impurities derived from trichloroacetamide wereremoved in this process. Acidification of the alkaline aqueous fractionfollowed by extraction of the methylene chloride confirmed that verylittle epoxymexrenone was lost in this operation.

The methylene chloride phase was washed once with 0.1 N hydrochloricacid (61 mL) for 15 min. under 250 rpm agitation at a temperature in therange 15-25° C. The layers were then allowed to separate and the lowermethylene chloride layer removed and washed again with 10% by weightaqueous sodium chloride (61 mL) for 15 min at 250 rpm at a temperaturein the range of 15-25° C. Again the layers were allowed to separate andthe organic layer removed. The organic layer was filtered through a padof Solkafloc and then evaporated to dryness under reduced pressure.Drying was completed with a water bath temperature of 65° C. Anoff-white solid (17.95 g) was obtained and submitted for HPLC assay.Epoxymexrenone assay was 66.05%. An adjusted molar yield for thereaction was 93.1%.

The product was dissolved in hot methyl ethyl ketone (189 mL) and theresulting solution was distilled at atmospheric pressure until 95 mL ofthe ketone solvent had been removed. The temperature was lowered to 50°C. as the product crystallized. Stirring was continued at 50° C. for 1hr. The temperature was then lowered to 20-250° C. and stirringcontinued for another 2 hrs. The solid was filtered and rinsed with MEK(24 mL) and the solid dried to a constant weight of 9.98 g, which byHPLC assay contain 93.63% epoxymexrenone. This product was re-dissolvedin hot MEK (106 mL) and the hot solution filtered through a 10 micronline filter under pressure. Another 18 mL of MEK was applied as a rinseand the filtered MEK solution distilled at atmospheric pressure until 53mL of solvent had been removed. The temperature was lowered to 50° C. asthe product crystallized; and stirring was continued at 50° C. for 1 hr.The temperature was then lowered to 20-25° C. and held at thattemperature while stirring was continued for another 2. hrs. The solidproduct was filtered and rinsed with MEK (18 mL). The solid product wasdried to a constant weight of 8.32 g which contained 99.6%epoxymexrenone per quantitative HPLC assay. The final loss on drying wasless than 1.0%. Overall yield of epoxymexrenone in accordance with thereaction and work up of this Example is 65.8%. This overall yieldreflected a reaction yield of 93%, an initial crystallization recoveryof 78.9%, and a recrystallization recovery of 89.5%.

EXAMPLE 48 Epoxidation of Formula IIA Using Toluene

The enester of Formula IIA was converted to eplerenone in the methodgenerally described in Example 46 except that toulene was used as thesolvent. The materials charged to the reactor included enester (2.7 g)trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7 g),hydrogen peroxide (17.0 g) and toulene (50 ml). The reaction was allowedto.exotherm to 28° C. and was complete in 4 hours. The resulting threephase mixture was cooled to 15° C., filtered, washed with water anddried in vacuo to yield 2.5 g of product.

EXAMPLE 49 Epoxidation of 9,11-Dienone

A compound designated XVIIA (compound XVII wherein —A—A— and —B—B— areboth —CH₂—CH₂—) (40.67 g) was dissolved in methylene chloride (250 mL)in a one liter 3 necked flask and cooled by ice salt mixture externally.Dipotassium phosphate (22.5 g), and trichloroacetonitrile (83.5 g) wereadded and mixture cooled to 2° C. after which 30% Hydrogen peroxide (200g) was slowly added over a period of 1 hour. The reaction mixture wasstirred at 12° for 8 hours and 14 hours at room temperature. A drop ofthe organic layer was taken and checked for any starting enone and wasfound to be <0.5%. Water (400 mL) was added, stirred for 15 min. andlayers separated. The organic layer was washed successively with 200 mLof potassium iodide (10%), 200 mL of sodium thiosulfate (10%) and 100 mLof saturated sodium bicarbonate solution separating layers each time.The organic layer was dried over anhydrous magnesium sulfate andconcentrated to yield crude epoxide (41 g). The product crystallizedfrom ethyl acetate:methylene chloride to give 14.9 g of pure material.

EXAMPLE 50 Epoxidation of Compound XVIIA Using M-chloroterbenzoic Acid

Compound XVIIA (18.0 g) was dissolved in 250 mL of methylene chlorideand cooled to 10° C. Under stirring solid m-chloroperbenzoic acid,(50-60% pure, 21.86 g) was added during 15 min. No rise in temperaturewas observed. The reaction mixture was stirred for 3 hours and checkedfor the presence of the dienone. The reaction mixture was treatedsuccessively with sodium sulfite solution (10%), sodium hydroxidesolution (0.5N), hydrochloric acid solution (5%) and finally with 50 mLof saturated brine solution. After drying with anhydrous magnesiumsulfate and evaporation, 17.64 g of the epoxide resulted and was useddirectly in the next step. The product was found to containBaeyer-Villiger oxidation product that had to be removed by triturationfrom ethyl acetate followed by crystallization from methylene chloride.On a 500 g scale, the precipitated m-chlorobenzoic acid was filteredfollowed by the usual work up.

EXAMPLE 51 Epoxidation of Compound XVIIA Using Trichloroacetamide

Compound XVIIA (2 g) was dissolved in 25 mL of methylene chloride.Trichloroacetamide (2 g), dipotassium phosphate (2 g) were added. Understirring at room temperature 30% hydrogen peroxide (10 mL) was added andstirring continued for 18 hours to yield the epoxide (1.63 g).Baeyer-Villiger product was not formed.

EXAMPLE 52

Potassium hydroxide (56.39 g; 1005.03 mmol; 3.00 eq.) was charged to a2000 mL flask and slurried with dimethylsulfoxide (750.0 mL) at ambienttemperature. A trienone corresponding to Formula XX (wherein R³ is H and—A—A— and —B—B— are each —CH₂—CH₂—) (100.00 g; 335.01 mmol; 1.00 eq.)was charged to the flask together with THF (956.0 mL).Trimethylsulfonium methylsulfate (126.14 g; 670.02 mmol; 2.00 eq.) wascharged to the flask and the resulting mixture heated at reflux, 80 to85° C. for 1 hr. Conversion to the 17-spirooxymethylene was checked byHPLC. THF approximately 1 L was stripped from the reaction mixture undervacuum after which water (460 mL) was charged over a 30 min. periodwhile the reaction mixture was cooled to 15° C. The resulting mixturewas filtered and the solid oxirane product washed twice with 200 mLaliquots of water. The product was observed to be highly crystalline andfiltration was readily carried out. The product was thereafter driedunder vacuum at 40° C. 104.6 g of the 3-methyl enol etherΔ-5,6,9,11,-17-oxirane steroid product was isolated.

EXAMPLE 53

Sodium ethoxide (41.94 g; 616.25 mmol; 1.90 eq.) was charged to a dry500 mL reactor under a nitrogen blanket. Ethanol (270.9 mL) was chargedto the reactor and the sodium methoxide slurried in the ethanol. Diethylmalonate (103.90 g; 648.68 mmol; 2.00 eq.) was charged to the slurryafter which the oxirane steroid prepared in the manner described inExample 52 (104.60 g; 324.34 mmol; 1.00 eq.) was added and the resultingmixture heated to reflux, i.e., 80 to 85° C. Heating was continued for 4hrs. arter which completion of the reaction was checked by HPLC. Water(337.86 mL) was is charged to the reaction mixture over a 30 min. periodwhile the mixture was being cooled to 15° C. Stirring was continued for30 min. and then the reaction slurry filtered producing a filter cakecomprising a fine amorphous powder. The filter cake was washed twicewith water (200 mL each) and thereafter dried at ambient temperatureunder vacuum. 133.8 g of the 3-methylenolether-Δ5,6,9,11,-17-spirolactone-21-methoxycarbonyl intermediate wasisolated.

EXAMPLE 54

The 3-methyl enolether-Δ5,6,9,11,-17-spirolactone-21-methoxycarbonylintermediate (Formula XVIII where R³ is H and —A—A— and —B—B— are each—CH₂—CH₂—; 133.80 g; 313.68 mmol; 1.00 eq., as produced in Example 53,was charged to the reactor together with sodium chloride (27.50 g;470.52 mmol; 1.50 eq.) dimethyl formamide (709 mL) and water (5 mL) werecharged to a 2000 mL reactor under agitation. The resulting mixture washeated to reflux, 138 to 142° C. for 3 hrs. after which the reactionmixture was checked for completion of the reaction by HPLC. Water wasthereafter added to the mixture over a 30 min. period while the mixturewas being cooled to 15° C. Agitation was continued for 30 min. afterwhich the reaction slurry was filtered recovering amorphous solidreaction product as a filter cake. The filter cake was washed twice (200mL aliquots of water) after which it was dried. The product3-methylenolether-17-spirolactone was dried yielding 91.6 g (82.3%yield; 96 area % assay).

EXAMPLE 55

The enol ether produced in accordance with Example 54 (91.60 g; 258.36mmol; 1.00 eq.) ethanol (250 mL) acetic acid (250 mL) and water (250 mL)were charged to a 2000 mL reactor and the resulting slurry heated toreflux for 2 hrs. Water (600 mL) was charged over a 30 min. period whilethe reaction mixture was being cooled to 15° C. The reaction slurry wasthereafter filtered and the filter cake washed twice with water (200 mLaliquots). The filter cake was then dried; 84.4 g of product 3-ketoΔ4,5,9,11,-17-spirolactone was isolated (compound of Formula XVII whereR³ is H and —A—A— and —B—B— are —CH₂—CH₂—; 95.9% yield).

EXAMPLE 56

Compound XVIIA (1 kg; 2.81 moles) was charged together with carbontetrachloride (3.2 L) to a 22 L 4-neck flask. N-bromo-succinamide (538g) was added to the mixture followed by acetonitrile (3.2 L). Theresulting mixture was heated to reflux and maintained at the 68° C.reflux temperature for approximately 3 hrs. producing a clear orangesolution. After 5 hrs. of heating, the solution turned dark. After 6hrs. the heat was removed and the reaction mixture was sampled. Thesolvent was stripped under vacuum and ethyl acetate (6 L) added to theresidue in the bottom of the still. The resultant mixture was stirredafter which a 5% sodium bicarbonate solution (4 L) was added and themixture stirred for 15 min. after which the phases were allowed tosettle. The aqueous layer was removed and saturated brine solution (4 L)introduced into the mixture which was then stirred for 15 min. Thephases were again separated and the organic layer stripped under vacuumproducing a thick slurry. Dimethylformamide (4 L) was then added andstripping continued to a pot temperature of 55° C. The still bottomswere allowed to stand overnight and DABCO (330 g) and lithium bromide(243 g) added. The mixture was then heated to 70° C. After one andone-half hrs. heating, a liquid chromatography sample was taken andafter 3.50 hrs. heating, additional DABCO (40 g) was added. After 4.5hrs. heating, water (4 L) was introduced and the resulting mixture wascooled to 15° C. The slurry was filtered and the cake washed with water(3 L) and dried on the filter overnight. The wet cake (978 g) wascharged back into the 22 L flask and dimethylformamide (7 L) added. Themixture thus produced was heated to 105° C. at which point the cake hadbeen entirely taken up into solution. The heat was removed and themixture in the flask was stirred and cooled. Ice water was applied tothe reactor jacket and the mixture within the reactor cooled to 14° C.and held for two hours. The resulting slurry was filtered and washedtwice with 2.5 L aliquots of water. The filter cake was dried undervacuum overnight. A light brown solid product 510 g was obtained.

EXAMPLE 57

To a 2 L 4-neck flask were charged: 9,11-epoxy canrenone as produced inExample 49, 50, or 51 (100.00 g; 282.1 mmol; 1.00 eq.),dimethylformamide (650.0 mL), lithium chloride (30.00 g; 707.7 mmol;2.51 eq.), and acetone cyanohydrin (72.04 g; 77.3 mL; 846.4 mmol; 3.00eq.). The resulting suspension was mechanically stirred and treated withtetramethyl guanidine (45.49 g; 49.6 mL; 395.0 mmol; 1.40 eq.). Thesystem was then filtered with a water cooled condenser and a dry icecondenser (filled with dry ice in acetone) to prevent escape of HCN. Thevent line from the dry ice condenser passed into a scrubber filled witha large excess of chlorine bleach. The mixture was heated to 80° C.

After 18 hrs., a dark reddish-brown solution was obtained which wascooled to room temperature with stirring. During the cooling process,nitrogen was sparged into the solution to remove residual HCN with thevent line being passed into bleach in the scrubber. After two hrs. thesolution was treated with acetic acid (72 g) and stirred for 30 min. Thecrude mixture was then poured into ice water (2 L) with stirring. Thestirred suspension was further treated with 10% aqueous HCl (400 mL) andstirred for 1 hr. Then the mixture was filtered to give a dark brick-redsolid (73 g). The filtrate was placed in a 4 L separatory funnel andextracted with methylene chloride (3×800 mL); and the organic layerswere combined and back extracted with water (2×2 L). The methylenechloride solution was concentrated in vacuo to give 61 g of a dark redoil.

After the aqueous wash fractions were allowed to sit overnight, aconsiderable precipitate developed. This precipitate was collected byfiltration and determined to be pure product enamine (14.8 g).

After drying the original red solid (73 g) was analyzed by HPLC and itwas determined that the major component was the 9,11-epoxyenamine. HPLCfurther showed that enamine was the major component of the red oilobtained from methylene chloride workup. Calculated molar yield ofenamine was 46%.

EXAMPLE 58

9,11-epoxyenamine (4.600 g; 0.011261 mol; 1.00 eq.) as prepared inaccordance with Example 57 was introduced into a 1000 mL round bottomflask. Methanol (300 mL) and 0.5% by weight aqueous HCl (192 mL) wereadded to the mixture which was thereafter refluxed for 17 hrs. Methanolwas thereafter removed under vacuum reducing the amount of material inthe still pot to 50 mL and causing a white precipitate to be formed.Water (100 mL) was added to the slurry which was thereafter filteredproducing a white solid cake which was washed three times with water.Yield of solid 9,11-epoxydiketone product was 3.747 g (81.3%).

EXAMPLE 59

The epoxydiketone prepared in accordance with Example 58 (200 mg; 0.49mmol) was suspended in methanol (3 mL) and1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) added to the mixture. Uponheating under reflux for 24 hrs. the mixture became homogeneous. It wasthen concentrated to dryness at 30° C. on a rotary evaporator and theresidue partitioned between methylene chloride and 3.0 N HCl.Concentration of the organic phase yielded a yellow solid (193 mg) whichwas determined to be 22% by weight epoxy mexrenone. The yield was 20%.

EXAMPLE 60

To 100 mg of the diketone suspended in 1.5 mL of methanol was added 10microliters (0.18 eq) of a 25% (w/w) solution of sodium methoxide inmethanol. The solution was heated to reflux. After 30 min. no diketoneremained and the 5-cyanoester was present. To the mixture was added 46microliters of 25% (w/w) sodium methanol solution in methanol. Themixture was heated at reflux for 23 hours at which time the majorproduct was eplerenone as judged by HPLC.

EXAMPLE 61

To 2 g of the diketone suspended in 30 ml of dry methanol was added 0.34mL of triethylamine. The suspension was heated at reflux for 4.5 hours.The mixture was stirred at 25° C. for 16 hours. The resulting suspensionwas filtered to give 1.3 g of the 5-cyanoester as a white solid.

To 6.6 g of the diketone suspended in 80 mL of methanol was added 2.8 mLof triethylamine. The mixture was heated at reflux for 4 hours and wasstirred at 25× for 88 hours during which time the product crystallizedfrom solution. Filtration followed by a methanol wash gave 5.8 g of thecyanoester as a white powder. The material was recrystallized fromchloroform/methanol to give 3.1 g of crystalline material which washomogeneous by HPLC.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above compositions and processeswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

What is claimed is:
 1. A compound of Formula IX:

wherein: R¹ represents an alpha-oriented lower alkoxycarbonyl orhydroxycarbonyl radical, and —A—A— represents the group —CHR⁴—CHR⁵— or—CR⁴═CR⁵—, R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy,hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, and aryloxy, and—B—B— represents the group —CHR⁶—CHR⁷— or an alpha- or beta-orientedgroup:

 where R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl,hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano, andaryloxy, and R⁸ and R⁹ are independently selected from the groupconsisting of hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl,alkoxyalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, acyloxyalkyl,cyano and aryloxy, or R⁸ and R⁹ together, comprise a carbocyclic orheterocyclic ring structure, or R⁸ and R⁹ together with R⁶ or R⁷comprise a carbocyclic or heterocyclic ring structure fused to thepentacyclic D ring.
 2. A compound of claim 1 wherein the compoundcorresponds to Formula IXA:

wherein: R¹ represents an alpha-oriented lower alkoxycarbonyl radical,—A—A— represents the group —CH₂—CH₂— or —CH═CH—, —B—B— represents thegroup —CH₂—CH₂— or an alpha- or beta-oriented group:

X represents two hydrogen atoms or oxo, Y¹ and Y² together represent theoxygen bridge —O—, or Y¹ represents hydroxy, and Y² represents hydroxy,lower alkoxy or, if X represents H₂, also lower alkanoyloxy, and saltsof compounds in which X represents oxo and Y² represents hydroxy.
 3. Acompound of claim 2 wherein: —A—A— represents the group —CH₂—CH₂—, and—B—B— represents the group —CH₂—CH₂—.
 4. A compound of claim 1 havingthe structural formula: